The purpose of this study was to determine the effect of sublethal concentrations of nitrofurantoin, ciprofloxacin, and trimethoprim on biofilm formation in 57 uropathogenic Escherichia coli strains (UPEC). The minimum inhibitory concentration of nitrofurantoin, ciprofloxacin, and trimethoprim was determined and the biofilm formation for each isolate with and without sub-lethal concentrations of each antibiotic was then quantified. The statistical significance of changes in biofilm formation was ascertained by way of a Dunnett’s test. A total of 22.8% of strains were induced to form stronger biofilms by nitrofurantoin, 12% by ciprofloxacin, and 19% by trimethoprim; conversely 36.8% of strains had inhibited biofilm formation with nitrofurantoin, 52.6% with ciprofloxacin, and 38.5% with trimethoprim. A key finding was that even in cases where the isolate was resistant to an antibiotic as defined by EUCAST, many were induced to form a stronger biofilm when grown with sub-MIC concentrations of antibiotics, especially trimethoprim, where six of the 22 trimethoprim resistant strains were induced to form stronger biofilms. These findings suggest that the use of empirical treatment with trimethoprim without first establishing susceptibility may in fact potentiate infection in cases where a patient who is suffering from a urinary tract infection (UTI) caused by trimethoprim resistant UPEC is administered trimethoprim. This emphasizes the need for laboratory-guided treatment of UTI.