I’ve done this several times with syslinux, and forget how each time. This time however I used a different method: have one FreeDOS partition, and another partition with your files on it. I found this much less error-prone than getting syslinux to correctly boot FreeDOS and see the files that you actually want to run (e.g. a BIOS update).

Here’s how:

1. Download FD12LITE.

2. Extract the img from FD12LITE:

   $ unzip -x FD12LITE.zip FD12LITE.img
   

3. Write that image to your USB stick (standard disclaimer: make sure sdb is the right device, and note that this overwrites all other data on the disk):

   # pv -rb FD12LITE.img | dd bs=1M oflag=direct,sync of=/dev/sdb
   

4. Use gparted to make a second FAT16 partition:

   # gparted /dev/sdb
   

   Use the largest size that will accomodate the target files.

5. Put what you need on there.

   # mount /dev/sdb2 /mnt/
   # cp <files> /mnt/
   # umount /mnt/
   

6. Boot from the USB stick. Select your language, and pick “No - Return To DOS”.

7. Switch to D:, and see your files with dir.

At any step, you can test with qemu-system-x86_64 -hda /dev/sdb.