An adenosine-5’-triphosphate (ATP) bioluminescence method was developed for detecting microbial activity in indoor air. This method was compared with the traditional method of collecting, culturing and counting CFUs. A comparison of the results showed that ATP bioluminescence, expressed as RLUs, was moderately correlated with the entire set of CFU counts (r = 0.607), and that the correlation improved to r = 0.963 (p value < 0.001) when CFU outliers were removed from the calculations. The ATP bioluminescence method was applied at four different sites; a hospital Chinese medicine diagnostic room, a library, a government office, and a railway station lobby. The results showed that microbial activity was far higher in the railway station lobby than at the other three sites, and this is believed to be due to the higher volume and density of people in this space. At all four sites, higher microbial activity was linked to indoor plants, garbage cans, shoe racks, and furnished waiting areas. PCA of the data showed that microbial activity in the Chinese medicine diagnostic room was closely related to room temperature and humidity, and hence lowering the latter can reduce the potential for microbial activity at this site. At all four sites, no correlation was found between microbial activity and airborne pollutants. The ATP bioluminescence method was applied for the rapid evaluation of room disinfection using chloride dioxide, and results showed that twenty minutes after spraying with 100 ppm ClO2, microbial activity was reduced to 38.7% of its original level. ATP bioluminescence is simpler, easier to operate, and more cost-effective than the conventional microbial culture method of evaluating microbial load. The results obtained in this research confirm that the proposed ATP bioluminescence technique is capable of instantaneously detecting microbial activity in an indoor environment. Moreover, this approach can be used for on-line evaluation of room disinfection efficiency.