Antimicrobials effective against meticillin-resistant staphylococci are limited. Mupirocin is a topical antimicrobial used to treat bacterial skin infections. Novobiocin is an oral antimicrobial approved for treatment of staphylococcal upper respiratory infections in dogs. This study reports the in vitro activity of mupirocin and novobiocin on meticillin-susceptible (MSS) and resistant staphylococci (MRS) from healthy dogs and dogs with superficial pyoderma. Staphylococci were isolated from skin swabs at four sites on healthy dogs and from lesions on dogs with superficial pyoderma. Staphylococci were identified by morphology and by catalase and coagulase testing. Speciation and susceptibility testing were performed by the Dade Microscan (W. Sacramento, CA, USA). Meticillin resistance was confirmed by an oxacillin screen plate. Novobiocin and mupirocin susceptibilities were tested by disc diffusion. Staphylococci were cultured from 61 healthy dogs (17 MRS and 44 MSS) and 30 dogs with pyoderma (15 MRS and 15 MSS), with higher proportions of MRS isolates in dogs with pyoderma (P=0.038; χ(2) test). For mupirocin, 79.5% (35 of 44) MSS and 82.3% (14 of 17) MRS isolates from healthy dogs, and 100% (15 of 15) MSS and 86.6% (13 of 15) MRS isolates from dogs with pyoderma were susceptible (MSS, P=0.094; MRS, P=1.0; Fisher's exact test). For novobiocin, 95.4% (42 of 44) MSS and 52.9% (nine of 17) MRS isolates from healthy dogs and 93.3% (14 of 15) MSS and 80% (12 of 15) MRS isolates from dogs with pyoderma were susceptible (MSS, P=1.0; MRS, P=0.148; Fisher's exact test).