Can I regenerate the EFT file if the Single Touch Payroll (STP) has already been sent off last week?

Yes, you can safely regenerate the EFT file even though the Single Touch Payroll (STP) has already been sent off last week. There won't be any issues with the system if you follow these steps again. Re-running these steps won't cause any problems and will allow you to proceed with this week's pay run without issues.