This study investigates the prevalence of Pseudomonas aeruginosa and Acanthamoeba spp. co-infection in dust from public parks in Eastern Thailand, a region with substantial air pollution from industrial activities. 336 dust samples collected from seven provinces were analyzed using microscopy and PCR to detect Acanthamoeba spp., with an overall prevalence of 22.32%. Co-infection with P. aeruginosa was confirmed in 43 samples (57.33%). Prachinburi province had the highest co-infection rate (75%), suggesting a potential link between environmental factors and pathogen prevalence. Analysis revealed a significant association between temperature and co-infection rates (p = 0.02), supporting previous findings on the impact of warmer climates on pathogen survival. Humidity, PM2.5, and PM10 levels did not significantly correlate with co-infection rates. These findings underscore the public health implications of dust as a reservoir for pathogenic microorganisms, particularly in areas with high pollution.