What is the purpose or historical significance of /usr/64, if any, on OS X? I have an old build script that makes mention (perhaps erroneously) of /usr/64 for things like library search paths when building on OS X.

This directory doesn't seem to be populated, or even exist, on any modern OS X that I can find, and I'd like to remove these references if that they are truly stale.

Was /usr/64 a real thing on OS X at one point? If so, what was it used for, and with what release did it cease to be relevant?
/usr/64 never existed in OS X, go ahead and remove it.

There was, however, a directory called "lib64" (or /usr/local/lib64), which offset /usr/lib. This was found on some Linux operating systems back some years ago, when the transition from 32bit to 64bit was still in its infancy. It may still be used today as a remnant of that era. Microsoft Windows also had a similar structure, separating 32bit from 64bit.

OS X has never used such a separation of libraries or even executables.