Bioaerosols, a significant yet underexplored component of atmospheric particulate matter, pose substantial public health risks, particularly in regions with poor air quality. This study investigates the composition of bioaerosols in public spaces, specifically two interstate motor parks and two marketplaces in Osun State, Nigeria, over six months. Air samples were collected, and bacterial and fungal species were identified, with a focus on pathogenic organisms. The results revealed the presence of well-known pathogens, including Staphylococcus aureus, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Aspergillus sp., and Fusarium sp., which are associated with respiratory and gastrointestinal infections, as well as antimicrobial resistance. Site-specific differences in microbial diversity were observed, with higher bacterial diversity in motor parks and greater fungal occurrence in marketplaces, influenced by environmental factors such as waste management. The findings highlight the urgent need for microbial air quality monitoring in public spaces, alongside improved sanitation practices. This study provides critical insights into the public health risks posed by bioaerosols and calls for both local and global interventions to mitigate the impact of airborne pathogens in urban environments.