The aim of this study was to evaluate the effects of human serum albumin (HSA), ibuprofen sodium (IBU) and N-acetyl-L-cysteine (NAC) against biofilm formation by seven biofilm-producing strains of Escherichia coli. Biofilm formation was studied using polystyrene microtitre plates in static conditions. The impact of the three compounds on bacterial growth and biofilm formation was tested by applying each compound in solution and as pre-treatment (coating) of polystyrene wells. When studied in solution, the minimum biofilm inhibitory concentrations of HSA, IBU and NAC were 8 mg/L (all strains), 2-125 mg/L (five strains) and 30-125 mg/L (five strains), respectively. Pre-treatment of polystyrene plates with HSA at 8 and 32,000 mg/L significantly reduced biofilm formation by all strains, whereas coating with 125 mg/L IBU and 1000 mg/L NAC did not. When HSA at 8 and 32,000 mg/L was combined with either 125 mg/L IBU or 1000 mg/L NAC in pre-treatment assays, more potent inhibition of biofilm was observed for some strains. Our results suggest that biofilm formation by E. coli may be prevented by coating medical devices with HSA alone or in combination with IBU or NAC. In addition, IBU and NAC could be useful in the treatment of urinary tract infections caused by E. coli due to their inhibitory effect on both bacterial growth and biofilm formation.