Best bet would be to switch the cable connector between the 3.5 and 5.25 drive

Some motherboards + BIOS allow "swap A -B" for floppies, would be listed under the Floppy portion if your board BIOS supports it.
Otherwise just do as weedeewee says......easier than messing with the drive jumpers (usually have to remove the drives, specially 5.25" to alter any jumpers.)

Just format an empty disk in drive A with VgaCopy.
It puts a bootsector on that disk that allows you to swap drives B and A and to boot from the disk in the other drive.

I tried to disconnect 5.25 drive in hope that it would pick up 3.5 drive as A:, but did not work that way. Also thought of swapping cables, but they are different kinds. 3.5 looks like sata, but 5.25 uses another type of connector. Something that goes over pcb, not classic pins. Ending on 5.25 drive looks more like pci card. Don't know how to call it, my knowledge of tech this old is limited.

There are adapters and some cables have both types for both drives.
But have you considered the trick I have suggested?

VgaCopy solution sounds great, but I don't have any other device to put anything on 5.25 floppy. And system also lacks any bootable drive. Maybe USB-SATA cable would do it? Just to put something bootable on C:?

So you cannot make a bootable disk for your 5.25" drive. Maybe you can ask a friend?

What kind of HDD do you have? Which error message do you get when booting from HDD? How is your HDD registered in BIOS (auto-detection)? Is there an operating system on your second HDD?
Perhaps you have a USB-PATA adapter to check the contents.
USB and SATA will most likely not help in booting issues on a 486.

Some I/O cards have an A/B swap jumper also. You could stick your turbo switch on it if you're not using your turbo switch.

Sucho wrote on 2021-12-12, 22:59:

I tried to disconnect 5.25 drive in hope that it would pick up 3.5 drive as A:, but did not work that way. Also thought of swapping cables, but they are different kinds. 3.5 looks like sata, but 5.25 uses another type of connector. Something that goes over pcb, not classic pins. Ending on 5.25 drive looks more like pci card. Don't know how to call it, my knowledge of tech this old is limited.

Yes there are different types of floppy cables and connectors. The best (called Universal) have two for 5.25" and two for 3.5" floppies so that you can change the drives from A to B easily.
The connector type for 5.25" floppies is a card edge type connector. The 3.5" floppy for a PC should have a 34 pin IDC connector like a standard IDE drive cable but with fewer pins.
The part that looks like a SATA cable is probably the 3.5" floppy power cable IF you have a standard 486 type computer....if not then you have an odd computer.
What brand of 486 computer is it ?
If your cable does not look like one of pictured below PLEASE take a picture of it and post it here. Some computers (some Compaq as example) use a non standard floppy cable.

@Disruptor
No friends dabble in this kind of stuff.
This is the C: HDD.
This is the D: HDD. But was never set up properly. Could see only like 50MB of it.
Battery is dead so I am using auto-detection in bios on every startup.
Funny thing is I actually installed Windows 3.0 on D: prior to losing original ms-dos on C:. Its contents are literally D:\Windows and D:\Games

@BitWrangler
This is the I/O card.
Please let me know if you find anything useful there! I am not sure what I am looking for.

@Horun
Thank you so much for taking the time to explain! My cable seems to be set on ruining my day. It has single 5.25 at the end of it and both 5.25 and 3.5 in the middle.
Yes sorry for the confusion, socketed intel 486dx2-50 and this should be the motherboard.
Could not find any brand on it. I can imagine there used to be a sticker, but that would be long gone.
Also there seems to be some disparity in motherboard and case. Some buttons on the front panel are not connected to the motherboard. For example turbo button requires 3 pins, but motherboard has only 2 pins labeled as turbo.

Edit: added info about case

https://www.pcorner.com/list/UTILITY/BOOT_B.ZIP/INFO/

@Jo22 Cool stuff, but I have no means of creating such 5.25 floppy. My only machine with such drive is this one, and right now can not boot into anything. Do not have any bootable 5.25 floppy, and ms-dos on C: got deleted in.. Well.. Space freeing accident - let's call it that.

With a bit of care, you might be able to unclip the 3.5" connector from the middle of the cable (B:), and then press fit it on somewhere after the twist (A:). Or get a new 34-way IDC connector to press on. I've done something similar to change the spacing of the connectors on a 40-way HDD cable.

Sucho wrote on 2021-12-13, 09:29:

This is the I/O card.
Please let me know if you find anything useful there! I am not sure what I am looking for.

Looks like it doesn't have one. Should be "A/B Swap" or something like that.

as Horun mentioned, you could mess with the jumpers on the floppy drives (if there are any), or just the 3.5 drive and leave the 5.25 drive off. that way you won't need to displace the connector, or find another cable or...
you'll just need to find the info on which jumper to move. if at all possible on the drive you have. 😀

It seems to me that drive sel for A: and B: are specific pins on the connector. Maybe I could use something like these to swap those pins in between I/O card and cable itself?

The thing is, its nearly impossible to get my hands on 5.25" ms-dos floppies.

Sucho wrote on 2021-12-13, 18:45:

It seems to me that drive sel for A: and B: are specific pins on the connector. Maybe I could use something like these to swap t […]
Show full quote

It seems to me that drive sel for A: and B: are specific pins on the connector. Maybe I could use something like these to swap those pins in between I/O card and cable itself?

yeah, you could do that.
You just need to wire it up with the same wires swapped as the cable is for the 5.25 connector.
Also you can hook it up but you will again have to leave the 5.25 drive off of the cable.

are you sure you don't have jumpers on your 3.5 floppy drive ?

weedeewee wrote on 2021-12-13, 18:51:

are you sure you don't have jumpers on your 3.5 floppy drive ?

I can see some there. But are in difficult to reach place. Maybe they are not meant to be touched? Also found one source stating that:

The cross-over of some of the wires in the floppy disk’s signals cable determines the drive’s physical address. The drive attach […]
Show full quote

The cross-over of some of the wires in the floppy disk’s signals cable determines the drive’s
physical address. The drive attached to the first connector is recognized as drive A, while
the drive attached to the second connector is recognized as drive B. Therefore there is no
need to make any jumper settings on the drive to determine its address.