The presence of bacteria and fungi in indoor environments can contribute to Sick Building Syndrome (SBS). In air-conditioned rooms, inadequate air circulation is the primary cause of microbial accumulation. To address this issue, the use of a simple exhaust fan is proposed as an effective alternative. This study aimed to determine the optimum operating time of an exhaust fan required to reduce microorganism concentrations below the recommended threshold values. A quasi-experimental design with repeated, non-randomized trials was conducted in four air-conditioned classrooms, with six repeated measurements per room. Air samples were collected using petri dishes containing Nutrient Agar (NA) and Potato Dextrose Agar (PDA), placed at five different points in each room. Results indicated that varying exhaust fan operating times significantly affected bacterial concentrations (p ≈ 0) and fungal concentrations (p = 0.023). To reduce bacterial levels below the threshold of 700 CFU/m³, a fan running time of 120 minutes was required, achieving 712.96 CFU/m³. In contrast, fungal concentrations were reduced below the threshold of 1000 CFU/m³ within just 30 minutes, reaching 548.35 CFU/m³.