Premeasured, clipped areas of skin on both sides of 30 adult dogs were prepared with povidone-iodine (PI), chlorhexidine gluconate (CG) with a saline rinse, or 4% CG with a 70% isopropyl alcohol rinse. Skin bacteria were quantified with Replicating Organism Detection and Counting (RODAC) plates and cultured for identification before, immediately after, and 1 hour after skin preparation. The percentages of bacterial reduction immediately and at hour 1 and the percentages of negative cultures, cultures with more than five colony-forming units (CFUs), and skin reactions were analyzed by analysis of variance and chi-square. The percentage of reduction in skin bacteria for all techniques was significant and comparable with that reported in humans. There were no significant differences between PI and CG results except that acute contact dermatitis was observed more frequently after skin preparation with PI. The authors conclude that for similar application times, PI and 4% CG rinsed with saline or 70% isopropyl alcohol are equally effective for up to 1 hour in the preoperative skin preparation of dogs.