A crackdown will be targeted at companies in Glasgow after the city was pinpointed as being a hotspot for illegal software.
Hundreds of businesses in the city escaped punishment during a 30-day amnesty last November on unlicensed software by the Business Software Alliance (BSA), an anti-counterfeit-software group.
But now the BSA is investigating 41 firms after a 20-fold increase in reports of alleged illegal software in the city during the amnesty.
The businesses, ranging from media companies to architects, face the threat of costly legal action and having to buy licensed software if they fail to convince the BSA they are operating legally.
Glasgow was targeted after the BSA received more reports of counterfeit software within businesses there than any other city across the UK, outside Greater London.
Further regional crackdowns are planned across the UK, with Manchester the next stop early this year.
Julie Strawson, chairman of the BSA in the UK, said in a statement: "We have just scratched the surface here; with anything like this you have to remember you can't just change the situation overnight. The main emphasis of the campaign was education around the risks of using illegal software."
She added: "Those businesses that have ignored warnings and are flouting software licensing laws will now face the consequences of failing to take this issue seriously."
Strawson said future crackdowns will pick up on the many creative companies inadvertently breaking the law by using unlicensed fonts.
Mohammad Sarwar, MP for Glasgow Central, said in a statement: "Glasgow's poor software piracy record threatens the city's economic stability as well as damaging its reputation."
During the amnesty, companies were given a chance to carry out a full software audit and given advice over their software management.
The BSA warned that using unlicensed software also risks loss or corruption of vital data and IT systems, citing research by the IDC that revealed one in three counterfeit products contain spyware, malware or viruses.