The complaint from the 41 online shopping rivals comes as Vestager's team investigates whether Google similarly favored its job-search business, Google for Jobs.
The letter has been signed by Axel Springer-owned Idealo, Polish price-comparison site Ceneo, Kelkoo and Foundem from the UK, and Czech site Heureka.
Foundem's founders Adam and Shivaun Raff were the lead complainant in the EU's Google Shopping investigation, but some of the signatories in the new letter are raising concerns for the first time.
Earlier this month Vestager said Google's auction-based response to the EU online shopping fine appeared not to have led to more traffic to its competitors.
Under Google's compliance proposal, comparison shopping sites can run product listing ads in Google Shopping results.
The companies say Google's attempt to present its measures as a compliance mechanism actually make matters worse for its competitors.
Google said the 41 companies that signed the letter to the European Commission represent only 6.8% of the total number in the CSS program.
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