Aims The aim of this study was to assess anti-biofilm and antimicrobial effects of auranofin, an anti-rheumatic agent, on uropathogenic Escherichia coli (UPEC) biofilm formation. Methods and Results The minimum inhibitory concentration and biofilm inhibition concentration of auranofin against UPEC ranged from 24 to 32 mu g ml(-1). Biofilm eradication concentration and XTT reduction concentration of auranofin were found to be at 112 mu g ml(-1). Confocal laser scanning microscopy results confirmed that biofilm was inhibited by auranofin. These results indicate that auranofin possesses potent anti-biofilm and antimicrobial activities against UPEC. Effects of auranofin on type 1 fimbriae gene (fimH) and response regulator gene (rpoS) to stress were explored using quantitative real time-polymerase chain reaction. In addition, combination of auranofin and tetracycline showed synergistic effect. Conclusions These data indicate that auranofin has inhibitory effect on biofilm formation and synergistic effect on UPEC infection when it is combined with tetracycline. Significance and Impact of the Study Our study strongly suggest that auranofin is a promising alternative anti-biofilm and antimicrobial agent to prevent UPEC biofilm formation in UTIs. Auranofin already approved for human use have the advantage of being able to be put into clinical use relatively quickly.