The EU's commissioner for competition, Margrethe Vestager, said Google has to "stop this behavior" in an interview with CNBC on Wednesday, after a record antitrust fine against the company.

"The thing that Google has to do now is of course to stop," Vestager told "Squawk on the Street." "This of course will free up the market to allow mobile manufacturers to use other Android systems."

Regulators hit the Alphabet unit with a $5 billion fine for abusing the dominance of its Android mobile operating system — by far the most popular smartphone OS in the world. The EU says Google pushed device makers to bundle Google apps like the Chrome web browser and Gmail, which harms competition. The European Commission, the EU's executive body, threatened additional fines if Google didn't put an end to illegal conduct within 90 days.

"They have products that we all like and like to use," Vestager said. “The only thing we don’t like is when they get to misuse their success and put in place illegal restrictions.”

Wednesday's fine is the largest ever issued to Google, dwarfing even the $2.7 billion penalty from the EU last year for favoring its shopping service over competitors. The company plans to appeal the ruling, according to a statement.

The commission is still investigating a third antitrust case against Google's search advertising service, AdSense.

"This is not about Apple, this is not about Android, this is about Google behavior — a behavior that's illegal for a dominant company because it's locking down competition and disabling innovation and choice that we would all like to enjoy," Vestager said.

She has emerged in recent years as one of the harshest critics of big tech, leading the effort out of Brussels to regulate U.S. technology firms.

"This is protecting European consumers," Vestager said. “This is our mission, these are our very basic values.”

— CNBC's Jillian D'Onfro, Ryan Browne and Ariel Shapiro contributed to this report.