Depends how well the software works.
I've had video games that went from unplayable (due to loading lag it was impossible to kill one of the major bosses as your character would die while the loading happened) to perfectly fine after a defrag.
Of course, in that case the file had mysteriously been split into 4000+ fragments...
Also forgot to mention, but I believe it only helps FAT and NTFS systems, EXT & some other Linux FS do not fragment files.
I can bend minds with my spoon.
A member of Team Graduation 2004/05!