Firms that let staff watch the World Cup on broadband could be fined £1,000 if they do not hold a TV licence.
Every live game on the BBC is also being shown on the website
The BBC is streaming more than 50 hours of World Cup football to UK internet users. England's next two games will only be available in audio online.
A TV Licensing spokesperson said: "We know exactly which unlicensed business premises to target."
More than five million UK workers are thought to have high-speed internet access at their desks.
A TV licence is required for any device that is "installed or used" for receiving television broadcasts.
"It doesn't matter how you're watching, if you are watching a live match you will need a licence," said a TV Licensing spokesperson.
The Federation of Small Businesses' Stephen Alambritis said: "We would urge all our members and other businesses to act on this advance warning and check they are correctly licensed."
The BBC can only show live video from matches broadcast on its television channels, which means England's next two games will only be available in audio online. TV coverage for those games is being provided by ITV.