The antimicrobial effects of red wine and its inherent components on oral microbiota were studied by using a 5-species biofilm model of the supragingival plaque that includes Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar. Microbiological analysis (CFU counting and confocal laser scanning microscopy) of the biofilms after the application of red wine, dealcoholized red wine, and red wine extract solutions spiked or not with grape seed and inactive dry yeast extracts showed that the solutions spiked with seed extract were effective against F. nucleatum, S. oralis and A. oris. Also, red wine and dealcoholized wine had an antimicrobial effect against F. nucleatum and S. oralis. Additional experiments showed almost complete and early degradation of flavan-3-ol precursors [(+)-catechin and procyanidin B2] when incubating biofilms with the red wine extract. To our knowledge, this is the first study of antimicrobial properties of wine in an oral biofilm model.