This study was performed to investigate the causes of bacterial brown blotch in Agaricus bisporus by analyzing environmental factors using sensors and growth factor characteristics. We found no direct correlation between the CO2 concentration and bacterial brown blotch among the evaluated cultivation environmental factors. However, maintaining the CO2 concentration at 500–2,000 ppm during the harvest period increased the hardness of the mushrooms. Ventilation for 40 min at dawn every day during this period helped prevent the development of bacterial brown blotch by reducing water condensation inside the grower. The incidence of browning disease was reduced to 1% when the medium layer humidity index was decreased to approximately 10 at harvest time and the leaf sensor moisture index was maintained at 430–490.