Climate change poses considerable challenges to poultry farming, as it affects the microclimate and air quality in enclosed poultry houses. This study investigated the influence of seasonal climate variations on the indoor microclimate of a commercial laying hen house. Over seven months, outdoor and indoor temperatures, relative humidity (RH%), air velocity (AV), and temperature-humidity index (THI) were systematically measured. The indoor temperatures closely mirrored the outdoor trends and significantly differed from the outdoor temperatures, except for April and May. The outdoor temperature significantly influenced the indoor front (Beta = 0.732) and back (Beta = 0.685) temperatures. Positive correlations were observed between the outdoor and indoor front (R² = 0.536) and back (R² = 0.470) temperatures. The indoor RH% was notably affected by the outdoor RH%, especially in warmer months. The outdoor RH% significantly predicted the indoor front (Beta = 0.463) and back (Beta = 0.427) RH%. Weak positive correlations existed between the outdoor and indoor front (R² = 0.214) and back (R² = 0.182) RH%. Indoor AV consistently lagged outdoor values, with no impact as a predictor of indoor front (P = 0.130) or back (P = 0.361) AV. The outdoor THI significantly influenced the indoor front (Beta = 0.774) and back (Beta = 0.751) THI. The indoor microbiological analysis revealed significant differences in the total colony count and the back-side total fungal count. In conclusion, these findings underscore the challenges posed by temperature and humidity fluctuations, highlighting the need for climate-responsive strategies to optimize indoor conditions for poultry production.