Everthing you say points to a software configuration issue - I know nothing about running a UC300ETH with Mach3 (the reason I bought my UC300ETH was to run away from Mach3). A quick look at the plugin for Mach3 shows the usual UC300ETH pin mappings - so I guess the plugin would normally save a configuration file somewhere within the Mach3 set-up. That would be my first port of call, but I expect you've looked at that already??

Other than that - I have no experience of a UC controller with Mach3 so will withdraw from this thread.