This study evaluated the indoor air quality in Brazilian universities by comparing thirty air-conditioned (AC) (n = 15) and naturally ventilated (NV) (n = 15) classrooms. The parameters of interest were indoor carbon dioxide (CO2), temperature, relative humidity (RH), wind speed, viable mold, and airborne dust levels. The NV rooms had larger concentration of mold than the AC rooms (1001.30 ± 125.16 and 367.00 ± 88.13 cfu/m3, respectively). The average indoor airborne dust concentration exceeded the Brazilian standards (<80 µg/m3) in both NV and AC classrooms. The levels of CO2 in the AC rooms were significantly different from the NV rooms (1433.62 ± 252.80 and 520.12 ± 37.25 ppm, respectively). The indoor air quality in Brazilian university classrooms affects the health of students. Therefore, indoor air pollution needs to be considered as an important public health problem.