In the last decade, atmospheric plasma has been used to treating bacterial infections in humans due to its bactericidal effects; however, its efficacy in dogs is unclear. This study evaluated the in vitro bactericidal efficacy of atmospheric plasma on Staphylococcus pseudinter- medius and Pseudomonas aeruginosa, two of the most important bacterial agents isolated from canine pyodermas. Three isolates each of S. pseudintermedius and P. aeruginosa obtained from dogs with pyoderma were subjected to atmospheric plasma. The isolates from the control group were not exposed to plasma, while those from the treatment groups were exposed to plasma for 15 (7.5 J/cm2), 30 (15 J/cm2), 60 (30 J/cm2), or 90 (45 J/cm2) seconds. After each treatment, a reduction in colony formation was observed. Bacterial viability was evaluated using the LIVE/ DEAD® BacLight™ Bacterial Viability Kit. The antibacterial effects were evaluated with Image J software and significance was assessed statistically in comparison to the control group. The bactericidal effect of atmospheric plasma against both bacteria increased significantly in a time-dependent manner. These results demonstrate the bactericidal capacity of atmospheric plasma, and suggest that it could serve as an alternative treatment method for canine pyoderma. Further studies are needed to evaluate the safety and efficacy of atmospheric plasma in dogs.