High levels of potentially toxic metals and microbes in the atmosphere, especially indoor air, may severely threaten human health. Therefore, the concentration and associated health risks of heavy metals (Cd, Cr, Pb, Cu, Fe, Mn, and Zn), biological pollutants, and their risk to human health were assessed using air condition (AC) filter dust samples. Samples were collected from five locations representing agricultural, industrial, and residential settings of the Eastern Province, Kingdom of Saudi Arabia. The levels of trace metals varied considerably among sampling areas, with the highest levels of Cr and Cd recorded in the industrial area sites, followed by the agricultural and residential sites. The highest levels of Pb and Fe were found in the agricultural area sites, followed by the industrial and residential area sites. Among all the metals Cd, Cr, and Pb, showed a considerable health risk through a dermal pathway, and health risks for children from indoor dust exposure were higher compared to adults. Among the sites, the highest hazard quotient for these metals was found for Al-Qatif industrial area sites, and among the metals, it was the highest for Cd. The cancer risk from the metals contained in AC filter dust was negligible. Samples collected from agricultural and industrial area sites were substantially contaminated with bacteria and fungi, respectively. Bacterial contaminants were mostly Gram-negative, with considerable antibiotic resistance and hemolytic activity. Thus, indoor air quality assessed by AC filter dust depicted that the trace heavy metals and microorganisms could pose a considerable health risk for long-term exposure. Furthermore, this study demonstrated that AC filter dust could be a unique and reliable test sample for indoor environment assessment.