This study aims to assess the airborne bioburden of rural and urban Portuguese Primary Health Care Centers (PHCC) using active and passive sampling methods and identify the potential differences in airborne microbiota between both environments. The highest total aerobic mesophilic bacterial load in indoor air were found in the Vaccination Room (448 CFU.m−3) in the Rural PHCC and in the Waiting Room (420 CFU.m−3) for Urban PHCC. The total coliforms contamination level in indoor air was detected only in the Cleaning Supplies Room (4 CFU.m−3) in the Urban PHCC. The most frequent bacteria genera identified was Micrococcus (21% Rural PHCC; 31% Urban PHCC). The surface samples showed a highest total aerobic mesophilic bacterial contamination in the Treatment Room (86 × 103 CFU.m−2) from the Rural PHCC and in the Front Office (200 × 103 CFU.m−2) from the Urban PHCC. The electrostatic dust cloth (EDC) samples showed a highest bacterial load in the Urban PHCC. Total aerobic mesophilic bacterial load in settled dust and in the Heating, Ventilating and Air Conditioning (HVAC) filter samples in the Urban PHCC (8 CFU.g−1 and 6 × 103 CFU.m−2) presented higher values compared with the Rural PHCC (1 CFU.g−1 and 2.5 × 103 CFU.m−2). Urban PHCC presented higher bacterial airborne contamination compared with the Rural PHCC for the majority of sampling sites, and when compared with the indoor air quality (IAQ) Portuguese legislation it was the Rural PHCC in two sampling places who did not comply with the established criteria.