Also DOS will not recognize files on anything but well-tempered FAT32 volumes usually, and the drive device needs to be MBR layout, not GPT. Plus the motherboard needs to support legacy CSM and have it enabled unless it's an old native-BIOS-only non-UEFI PC.
If you're using the FreeDOS ISO in Ventoy, maybe replacing it with a FreeDOS IMG that is a bitwise copy of a working "floppy" image would be a workaround?
Or add a second partition on your Ventoy stick for FreeDOS to occupy on its own and choose it like bare metal dual-boot style?
I actually still put MS-DOS on some sticks [0], haven't used FreeDOS in a number of years, but either way I do prefer for the stick to have a partition table and resemble a HDD, rather than the simpler non-partitioned floppy-simulating layout, even when they both work.
[0] People are still not disappointed by Tetris :)