Diet is one of the main factors that could affect quantitatively and qualitatively the stability of the gut microbiota. Polyphenols are abundantly present in the human diet and have an antimicrobial effect inducing selective changes in the microbiota composition, with potential beneficial effects for the human health. Our aim was to determine the human gut microbiota susceptibility toward wine polyphenols. Susceptibility to two commercial wine phenolic extracts (Vitaflavan(®) and Provinols™) was determined in isolates from fecal samples from 36 gastrointestinal healthy volunteers. To select the polyphenol-resistant isolates, feces were seeded in plates containing 1 mg/ml of phenolic extract. The minimal inhibitory concentration to polyphenols in the collected isolates was assessed by the agar dilution method. Overall results showed that Gram-negative isolates are most tolerant to the presence of both grape seed and red wine extracts. Furthermore, we purified to homogeneity the phenolic fractions by high-performance liquid chromatography (HPLC) to determine their antimicrobial effect and their influence on bacterial growth in four selected ATCC strains using the BioScreen apparatus. Results showed that the antimicrobial activity of the wine polyphenols is the result of the interaction of both the flavan-3-ol type and the bacteria. Bacterial Intraspecies differences in the phenolic susceptibility suggest the existence of polyphenol-resistant mechanisms that are uncharacterized as yet.