Biofilm formation can cause refractory urinary tract infections (UTIs) in dogs; however, minimum biofilm eradication concentrations (MBECs) of veterinary drugs against canine uropathogens remain to be investigated. In this study, the MBECs of orbifloxacin (OBFX), trimethoprim-sulfamethoxazole (TMS) and amoxicillin/clavulanate (ACV) over different time periods for treatment of canine uropathogenic Escherichia coli (n = 10) were determined. The MBECs of OBFX for other bacterial uropathogens, including Staphylococcus pseudintermedius (n = 5), Pseudomonas aeruginosa (n = 5), Klebsiella pneumoniae (n = 5) and Proteus mirabilis (n = 5) were also determined. Minimum inhibitory concentrations (MICs) were identified for all strains by broth microdilution, and MBECs were determined at 24, 72, and 168 hr using the Calgary biofilm method. The 24 hr MBECs of OBFX, TMS and ACV for the E. coli strains were significantly higher than the MICs (P < 0.05), and the 72 and 168 hr MBECs were significantly lower than those at 24 hr (P < 0.05). In addition, the 24 hr OBFX MBECs for the four other uropathogens were significantly higher than the corresponding MICs (P < 0.05). The 72 and/or 168 hr OBFX MBECs for S. pseudintermedius, K. pneumoniae and P. mirabilis were significantly lower than the 24 hr concentrations (P < 0.05), whereas for P. aeruginosa, no significant difference was found between any of the MBECs (P > 0.05). These data indicate that the administration period and uropathogenic bacterial species are important factors affecting the efficacy of OBFX treatment of biofilm-related UTIs in dogs.