Microbial aerosols cause various human and animal health problems, and thus their control is a major scientific and technological concern. The air quality within student cafeteria in Taiwan should comply with the guidelines laid down by the Taiwan Environmental Protection Agency. Accordingly, this study performed an experimental investigation into the efficiency of gaseous chlorine dioxide (ClO2) in disinfecting a local student cafeteria. The air quality before and after disinfection was evaluated in terms of the bioaerosol levels of bacteria and fungus, respectively. The average background levels of bacteria and fungus in the indoor area of the cafeteria prior to treatment were found to be 741.0 ± 311.4 and 799.9 ± 302.7 CFU/m3, respectively, during the semester and 381.7 ± 165.8 and 974.4 ± 294.6 CFU/m3, respectively, during vacation. Following a one-off application of gaseous ClO2, the average background levels of bacteria and fungus reduced to 661.0 ± 356.5 and 626.0 ± 265.1 CFU/m3, respectively, during the semester and 349.1 ± 183.2 and 853.4 ± 351 CFU/m3, respectively, during vacation. The disinfection efficiency of the ClO2 during the semester was reduced as the result of cooking, dishwashing, the passage of people, the ventilation system, and so on. Similarly, during vacation, the disinfection efficiency was reduced as the result of a greater accumulation of residual fungus due to a lack of regular cleaning. Therefore, the results obtained in this study suggest that a regular (i.e., daily) application of ClO2 is a more suitable protocol for maintaining an acceptable air quality in student cafeteria than a periodic, one-off application.