The prevalence of pandemics, such as COVID-19 transmitted through the respiratory system, is a serious global public health concern. Microbial contamination of face masks used by healthcare workers may vary depending on the degree of exposure to bioaerosol in various healthcare environments; however, data on this topic are limited. Therefore, in this study, we analyzed the microbial contamination of N95 masks used in hospital offices, wards, and outpatient settings. Total indoor airborne bacteria were also measured in healthcare environments. The samples isolated from N95 masks worn for 2, 4, or 6 h were incubated at a temperature of 35–37 °C or 25–28 °C for 24 h or 3–7 days, and colony-forming units were counted in chocolate agar, tryptic soy agar, and Sabouraud dextrose agar plates. Finally, microbial species were identified using the morphological analysis with Gram staining. The results showed that the three types of environments did not deviate from maintenance of standard indoor air quality. The numbers of bacteria in the masks worn in each environment differed, while the degree of contamination increased with mask-wearing time (p