This study investigated the impact of regular disinfection on the concentration of microbial aerosol and the distribution of microbial species in a university library and analysed the main sources of microorganisms. The microorganisms in the air were sampled by six-stage Andersen cascade impactors, and their diversity was explored based on ribosomal DNA site amplification and sequencing technology. The statistical analysis found that the average concentrations of bacteria and fungi in this library were 149 ± 36 CFU/m3 and 118 ± 33 CFU/m3, respectively. The concentration in the reading rooms, the hybrid rooms and the stack rooms decreased in succession. The disinfection effect was significant comparing the result with non-disinfected libraries reported in literature. The concentration ratio of indoor and outdoor microorganisms (I/O) was greater than 1, and the concentration of microbial aerosol was positively correlated with the concentration of CO2 and particulate matter. The results indicate that the microbial concentration was mainly from indoor occupants. The most prevalent microorganisms were Paenibacillus, Bacillus, Penicillium and Cladosporium. This species' distribution was not affected by disinfection measures. The median diameter of microbial aerosols in most rooms was less than 5 μm. The use of disinfectants did not affect the particle size distribution of microorganisms.