Microbes are vital components of atmospheric fine particle matter (PM2.5), and can have critical effects on human health (such as causing infectious diseases). Moreover, although China suffers from extensive concerns regarding human health issues related to air pollution, relatively few studies have focused on the characteristics of airborne microorganisms with respect to variations in air pollution, particularly under non-serious conditions. In this study, we used the modified fluorescein diacetate hydrolysis method to measure the seasonal microbial activity in PM2.5 during two levels of air pollution in Beijing. The mean microbial activity under excellent and good conditions (air quality index of < 100) was distinctly higher than that under slight and moderate conditions (101–200) during all four seasons. We further explored relationships between microbial activity and bacterial species richness and community diversity. The relative abundance of pathogenic bacteria was quite low overall, being highest in autumn (0.229%) and declining through winter (0.207%), summer (0.034%), and spring (0.022%), the same pattern observed for microbial activity. However, there was no statistical difference in the relative seasonal abundance of pathogens. Of the six types identified, streptococcus was most prevalent (0.23%). This study provides a reference and starting point for future research into the mostly unexplored field of pathogenic mechanisms during non-serious air pollution conditions.