"It imposes an unjustified fine and requires changes that will hurt thousands of European businesses by making it harder for them to make money," said Lee-Anne Mulholland, global head of regulatory affairs at Google.
"There's nothing anti-competitive in providing services for ad buyers and sellers, and there are more alternatives to our services than ever before."
US President Donald Trump also attacked the decision, saying in a post on social media it was "very unfair" and threatening to launch an investigation over European tech practices that could lead to tariffs.
"As I have said before, my Administration will NOT allow these discriminatory actions to stand," he wrote.
"The European Union must stop this practice against American Companies, IMMEDIATELY!"
Trump has repeatedly criticised the bloc's fines and enforcement actions against US tech firms in recent months, though the US government has brought its own lawsuits over Google's monopoly of the online ad market.
Earlier this week, the Commission denied reports it had delayed the announcement of Google's fine amid tensions over trade relations between the EU and the US.
In the Commission's decision on Friday, the Commission accused Google of "self-preferencing" its own technology above others.
As part of its findings, it said Google had intentionally boosted its own advertising exchange, AdX, over competing exchanges where ads are bought and sold in real-time.
Competitors and publishers faced higher costs and reduced revenues as a result, it said, claiming these may have been passed to consumers in the form of more expensive services.
The regulator has ordered the company to bring such practices to an end, as well as pay the nearly €3bn penalty.