Background: Computers and other electronic devices are a requisite aspect of people’s lives globally for multiple purposes, including education, entertainment, and communication. Objectives: This study investigated the bacterial contamination of desktop computer keyboards (as a reservoir of pathogens) in different departments of Mazandaran University of Medical Sciences, Sari, Northern Iran, from September 2018 to February 2019. Methods: In this descriptive cross-sectional study, samples were obtained from computer keyboards with sterile swabs and cultured on blood agar and eosin methylene blue agar. Standard microbiological methods were used to identify bacterial isolates. Then, the antimicrobial susceptibility testing of the isolates was performed using the Kirby-Bauer method based on the CLSI procedure. Results: In total, 58 bacterial strains were isolated from the collected samples. The isolates included 23 (39.7%) Staphylococcus epidermidis, 15 (25.9%) Staphylococcus aureus, 14 (24.1%) Bacillus spp., 3 (5.2%) Klebsiella pneumoniae, 2 (3.4%) Pseudomonas aeruginosa, and 1 (1.7%) Escherichia coli. All Gram-positive bacteria were resistant to penicillin, whereas the most resistance rate among Gram-negative bacteria was observed against ciprofloxacin, ceftazidime, and cephalothin. Conclusions: Due to the presence of opportunistic pathogens on computer keyboards, personal hygiene and periodic cleaning of keyboards with disinfectants is necessary to prevent the further spread of these bacteria.