Maintaining healthy indoor air quality (IAQ) in childcare settings is essential for infants and young children, as it directly impacts their early learning, development, and overall well-being. Given their vulnerability, continuous IAQ monitoring in these environments is crucial to ensuring a safe and supportive atmosphere. This study aimed to assess IAQ factors that may affect occupant health by measuring indoor concentrations of particulate matter (PM₁₀), selected gases such as carbon dioxide (CO₂) and formaldehyde (CH₂O), and thermal conditions including temperature and relative humidity. Additionally, airborne microorganism levels were analyzed, and potential environmental factors influencing microbial abundance were investigated in three childcare centers in Megrine, Tunisia, across three seasonal periods. Results revealed frequent occurrences of hygrothermal discomfort and elevated levels of CO₂, CH₂O, and PM₁₀, particularly in overcrowded classrooms with poor ventilation and heating. Pathogenic bacterial species, including Staphylococcus epidermidis, Staphylococcus haemolyticus, Bacillus cereus, and Bacillus licheniformis, were repeatedly detected. Significant correlations were found between bacterial abundance and environmental factors such as PM₁₀, CO₂ levels, temperature, and humidity. These findings provide valuable insights into IAQ dynamics in childcare environments, highlighting the need for improved ventilation and air quality management strategies to safeguard children's health and well-being.