along a similar vein of thinking...have you turned on some debugging output for CGI.pm and or checked your webserver logs? run the CGI from command line?..i think you can even provide the file upload parameter on command line. is the file upload form parameter being passed into the CGI for sure? sniff the network with wireshark? check webserver config is not pointing to older (incompatible) .dll/.exe for perl. also check the upgraded perl doesn't point to older modules somehow/somewhere.
i'm sure you know all these things to check..probably just stressed out too much. just remembered another useful thing: run Filemon utility and check the log, possibility of a file permission issue.
good luck.