The in vitro susceptibility of seven Escherichia coli biofilm-producing strains in their planktonic and biofilm-associated forms to amoxicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin, and ciprofloxacin was studied. Minimum inhibitory concentrations (MICs) were determined by the standard microdilution method and by the Alamar blue assay (providing the AB-MIC) at two levels of metabolic suppression and using standard and large inocula. Minimal biofilm inhibitory concentrations (AB-MBICs) on preformed biofilms on polystyrene plates were higher than the MICs and AB-MICs. Differences in magnitude depended on the antibiotic, strain, inoculum size, and the level of suppression of metabolism. Ciprofloxacin and gentamicin showed the greatest differences in the AB-MBIC as compared to AB-MIC. the possibility of antibiotic-resistant mutant selection within the biofilms was ruled out since bacteria recovered from the biofilm maintained the same MICs as before exposure to the antimicrobial agents. E. coli biofilms were much less sensitive than their planktonic counterparts.