Airborne transmission of pathogenic aerosols via human breath plays a major role in infectious disease outbreaks in indoor environments. Yet, their bioaerosol emission profiles are still not well quantified. Here, we first studied bioaerosol emission rates of human exhaled breath from 12 healthy subjects, and then evaluated the bioaerosol emissions when wearing two different respirators “Doctor masks” and N95 in a controlled environment (27 m3) using a bioaerosol sensor-ultraviolet aerodynamic particle spectrometer (UV-APS). The human bioaerosol contribution was further confirmed through classroom observation. The results showed that there was a peak around 1.5 µm for the fluorescent particles emitted from humans’ breath. For the controlled environment, the presence of 5 people without wearing masks increased bioaerosol concentration by 107% within 30 min at an average emission rate of 8.4 × 105 fluorescent particles person–1 hour–1 resulting from the occupancy. When wearing N95 masks or “Doctor masks”, bioaerosol increases were observed to be 81% or 31% for the controlled environment, respectively, lower compared to those without masks. In-classroom observation also showed a fluorescent particle concentration increase of about 50%. In all experiments, we observed a decline in PM number concentration. Bioaerosol emission from exhaled breath was calculated to account for about 17% of the increase in the controlled environment. The results here suggest the need for re-evaluating microbial aerosol exposure risks for medical sites that demand high levels of hygiene even while wearing a respirator.