Background and Objective: Acne is one of the medical problems and propionibacterium acnes is the pathogenic agent that has received a considerable attention. Different parts of Juglans regia have been shown to have antibacterial activity on many bacteria. Its leaves have antioxidant property and if it shows antibacterial effect on propionibacterium acnes, it might be an alternative drug for the treatment of this problem. Therefore, this study was aimed to determine the antibacterial effects of walnut leaves on propionibacterium acnes in vitro. Materials and Methods: In this experimental study, ethanolic extract of walnut leaves was examined for antibacterial activities on propionibacterium acnes using the disk-diffusion minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC) and macrodilution methods. Ampicillin (10µg) was used as a positive control. Results: Ethanolic extract of walnut leaves had an inhibitory effect on Propionibacterium acnes using the agar-diffusion and macrodilution methods. The most inhibition zone was 25 ± 1.7 mm in 150 mg/ml concentration. The minimum inhibitory concentration was 12.5 mg/ml while the minimum bactericidal concentration was 15 mg/ml. Conclusion: These findings showed that ethanolic extract of walnut leaves had antibacterial effect on Propionibacterium acnes and may be considered as an alternative drug for the treatment of external infections due to this microorganism.