Abstract. Boonman N, Chutrtong J, Wanna C, Boonsilp S, Chunchob S. 2022. Detection of Staphylococcus aureus from contact surfaces of public buses in Bangkok and metropolitan area, Thailand. Biodiversitas 23: 3395-3400. The purpose of present investigation was to determine the prevalence of different species of genus Staphylococcus on the contact surfaces of public buses in Bangkok and metropolitan area. A total of 180 samples were collected from handrails, seats, and window frames of each bus of 2 bus terminals, Bangkhen and Morchit, divided into 15 non-air-conditioned buses and 15 air-conditioned buses from each terminal. The samples were cultivated and identified by Gram’s stain and biochemical tests. The results showed that 76.7% from Bangkhen and 80% from Mochit were contaminated with Staphylococcus spp. Based on the type of vehicles, Staphylococcus spp. was found in 93.3% of air-conditioned buses and 63.3% of non-air-conditioned buses. Considering the sampling location, Staphylococcus spp. was found in 28.3% of handrails, 50% of seats and 51.7% of window frames. Four isolates of Staphylococcus aureus were discovered among 294 isolates in a total of Staphylococcus spp., including 3 isolates from Bangkhen and 1 isolate from Mochit. Only one isolate was contaminated on the non-air-conditioned bus, whereas 3 isolates were on the air-conditioned buses. Two isolates of them were found from seats and 2 isolates from window frames. Disk diffusion susceptibility test showed that 3 isolates of S. aureus resisted fusidic acid and fosfomycin. The results suggested that Staphylococcus spp. and S. aureus were prevalence in public transport systems which were a source of infection to the persons. Therefore, effectively controlling their distribution is necessary to reduce the risk of bacterial infections in public bus users.