Bacteria living in biofilms as dental plaque on tooth surfaces are generally more resistant to antimicrobial agents than bacteria in batch culture normally used for in vitro susceptibility testing. In order to compare the resistance of free-living and surface-grown oral bacteria, the MIC of Streptococcus sanguis 804 and ATCC 10556 to amoxicillin, doxycycline and chlorhexidine was determined by a broth dilution method. Subsequently, S. sanguis biofilms established in an in vitro flow model were perfused with the antimicrobial agents for 48 h at concentrations equal to and up to 500 times the MIC, and biofilm cell number was determined during this period. The antibiotics at the MIC did not affect the cell number of S. sanguis biofilms compared to the starting point, and only after 48 h at 500 times the MIC were the biofilm bacteria eliminated. At intermediate concentrations biofilm cell number gradually decreased. Chlorhexidine also gradually reduced biofilm cell number, but was inhibitory at concentrations closer to the MIC than was the case for the antibiotics. Thus S. sanguis in biofilms survived up to 500 times the MIC found in batch culture for up to 48 h.