Bioaerosols play a significant role in indoor air quality (IAQ) as they can be the cause of several health problems, including acute allergies and infectious diseases. This study aimed to characterize and compare the microbial air quality of air-conditioned (AC) and naturally ventilated (NV) office rooms in the Upper Silesia region of Poland. The bacterial samples were collected during the late spring season. Culturable bacteria were deposited on the nutrient media on Petri dishes to investigate the viable-culturable count (VCC) of bacteria and bacterial community structure using a Biolog GEN III system. In total, 12 species of bacteria were identified, with the most isolated Macrococcus equipercicus, Micrococcus luteus D, Staphylococcus xylosus (indoor), and Bacillus species (outdoor). The indoor mean concentrations of bacterial aerosol ranged from 102 to 103 CFU m−3, below Polish proposals for threshold limit standards in office buildings. The indoor-to-outdoor (I/O) ratios indicated that studied air pollutants in the office rooms originated from the indoor air. These results, together with community composition of bacteria, indicate that most of the bacteria present in the studied office building were relatively fresh and of human origin. Multi-antibiotic resistance (MAR) tests showed that the most antibiotic-resistant features were present in Macrococcus species. The office building exposure dose (OBED) and mass median aerodynamic diameter (MMAD) of bacteria aerosol were estimated. The highest value of OBED over the study period was obtained for staff working in offices with natural ventilation (141 CFU kg−1), in contrast to the value for staff working in offices equipped with air conditioning (about 100 CFU kg−1). The MMAD of viable airborne bacteria was higher in AC offices (2.4 μm) than in NV offices (2.2 μm).