Bioaerosols are a major source of pollution in indoor environments, where people spend approximately 90% of their time, and the microorganisms adhered to PM2.5 adversely affect human health. However, most research has focused on the concentration of these aerosols and the factors that influence it rather than the correlation between microbial activity and air quality. Thus, this study used a modified technique of fluorescein diacetate (FDA) hydrolysis to evaluate the activity of microorganisms in the PM2.5 during three seasons (summer, autumn and winter) in Beijing. 0.155–5.388 ng m–3 and 0.091–5.740 ng m–3 of sodium fluorescein, a marker of microbial activity, were measured indoors and outdoors, respectively; thus, no significant difference in concentration between the two environments was detected, but the indoor activity was affected by outdoor conditions to an extent. The most active season was autumn, followed by winter and summer. Furthermore, the highest activity in summer and autumn was observed during conditions of excellent air quality, and in summer, the activity during conditions of good air quality also obviously exceeded that during conditions of slight pollution. Additionally, the microbial activity in a room varied according to the room’s ventilation (or lack thereof), suggesting a strong association between these parameters. In general, when the air quality was excellent, 20 minutes of ventilation achieved the optimal air exchange, but this duration should be reduced during polluted conditions. Our results provide new insights into evaluating the indoor air quality based on the microbial activity.