A higher concentration of airborne micro-organisms and toxic trace metals in the air is a potential risk for human and environmental health. Suspended dust and microbes in the air are the main inducers of respiratory symptoms. This study aimed to estimate and characterize the microflora and heavy metal contaminants in the car cabin filters (CAFs) dust of Lahore, Pakistan. Moreover, toxicological characterization of the dust was done using relevant cell culture of Human Lung Alveolar Epithelial Cells A-549 through MTT assay. Culturable bacteria and fungi populations were quantified using the standard spread plate method and heavy metals lead, chromium, and cadmium concentrations were checked through atomic absorption spectroscopy. Kirby-Bauer disk diffusion method was used to determine the antibiotic resistance profile of the isolated bacterial species. The mean concentration of culturable bacteria and fungi was 4X10⁶ and 5X10⁵ CFU/g of dust respectively. Both Gram-Positive and Gram-Negative bacterial species with substantial antibiotic resistance were present in the dust. The bacteria present in the CAFs dust were resistant to ampicillin, cefoxitin and gentamycin while susceptible to ciprofloxacin and gentamycin. The heavy metal concentrations were as high as 273 mg/kg for Pb, 192 mg/kg for Cr and 11.4 mg/kg for Cd. The cell viability decreased significantly with the increase in dust concentration from 100 to 400 μg/mL. The findings of this study will be useful to policymakers and health care professionals to develop and implement effective interventions to prevent adverse health impacts of bioaerosols and trace metals on the local population.