The antimicrobial activity of twenty two synthetic flavonoids is reported. Among them three 3-arylideneflavanones, 2b, 2c, and 2i, were shown to be highly active against Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis reference strains, with MIC (minimal inhibitory concentration) values ranging from 4.68 microg/ml (14.3 microM) to 37.5 microg/ml (119.7 microM). The synergy of oxacillin and vancomycin with 2c, evaluated as fractional inhibitory concentration index (FICI) was shown (against planktonic culture of S. aureus A3 and E. faecium 138/09 clinical strains). The presence of 2c in the culture medium diminished the initial adhesion of bacteria to an abiotic surface. Such an effect resulted in a decrease in biofilm formation during prolonged culture. Unfortunately, 2e failed to eradicate the S. aureus mature biofilm which was already preformed, however, decreased the number of live biofilm cells. The biofilm of E. faecalis was more susceptible to the action of 3-arylideneflavanone 2c than the S. aureus biofilm. The finding that 3-arylideneflavanones are lipophilic, cause bacterial aggregation, and influence the integrity of membranes making them permeable to SYTO 9/propidium iodide dyes may implicate the cytoplasmic membrane as a target site for these compounds activity.