To ensure the proper quality of the air and simultaneously improve the comfort of professional drivers or travellers, the cars are fit with air-conditioning (AC) systems. The AC installations, however, quite often create suitable conditions for the development of microbiological agents and become an active emission source of harmful bioaerosols. This study was carried out in 6 municipal buses in large city located in central Poland. In studied vehicles, ozonisation or manual application of chemical disinfectant in a spray form were used for disinfection of AC bus systems. The concentrations of bacteria and fungi in the air collected in buses before the service of their AC installations were 4.3×102 CFU/m3 and 2.4×102 CFU/m3, respectively. After AC service using both tested disinfection methods, the decrease in microbial concentrations in the air of municipal buses was observed. The highest effectiveness of AC cleaning was noted when the chemical disinfectant in a spray form was used. This study revealed that to eliminate microbiological contamination and, by that, decrease the exposure of both drivers and passengers to harmful microbial contaminants, the chemical disinfection of bus AC system should be performed at regular basis.