The efficacy of 3% chloroxylenol (PCMX) or 4% chlorhexidine gluconate (CG) for preoperative skin preparation was assessed in 100 dogs undergoing clean or clean-contaminated surgical procedures. Replication Organism Detection and Counting (RODAC) plates were used to quantify skin bacteria colony forming units (CFU) at the operative site before and after skin preparation and immediately postoperatively. Reduction of CFU after skin preparation and immediately postoperatively was significant for each agent. However, CFU levels were significantly lower in the CG group than in the PCMX group after surgical preparation, regardless of initial CFU numbers. No significant difference in CFU counts was observed between antiseptic groups postoperatively. Within-group comparisons showed PCMX to be significantly less efficacious when the prescrub CFU number was greater than 1,000. Bacterial reduction was similar in the CG group regardless of prescrub CFU levels. The number of negative cultures after skin preparation was significantly greater with CG than with PCMX. Chlorhexidine gluconate also had fewer cultures with heavy bacterial growth (> 5 CFUs) after surgical preparation. There was no significant difference between antiseptics in the number of negative cultures or cultures with more than 5 CFUs immediately after surgery. The number of skin reactions and postoperative wound infections that occurred with each technique were similar. Three percent PCMX, as used in this study, was less effective than 4% CG in its immediate antimicrobial activity, however, this difference was not associated with an increased wound infection rate.