Research has been focused on the health hazards of ambient PM2.5 related to humans. Many PM2.5 toxicity assessments using in vitro studies have focused on PM2.5-bounded hazardous pollutants. However, PM2.5 toxicity assessment by in vivo studies allow for better observation of the overall effects of PM2.5 exposure on entire organisms, making in vivo PM2.5 toxicity assessment relevant. The toxic effects of outdoor PM2.5, collected from National Pingtung University of Science and Technology (NPUST) and Linluo Junior High School (LJHS), Pingtung, Taiwan, on nematode Caenorhabditis elegans (C. elegans) were investigated. PM2.5 from NPUST and LJHS were found to be 4.5 and 2.5 μg Nm–3, respectively, which did not meet the standard. This levels of PM2.5 in Taiwan. For acute toxicity, no significant PM2.5 lethality on C. elegans was observed between NPUST and LJHS. PM2.5 from NPUST exhibited greater toxicity to lifespan (ageing), locomotion (head thrash), and reproduction (brood size) in the C. elegans animal models than that from LJHS; therefore, adverse effects could be correlated with PM2.5 concentrations. Prolonged exposure to PM2.5 led to more severe toxicity in nematodes as compared to acute exposure. In conclusion, this study suggests that the long-term adverse effects of ambient PM2.5 on environmental organisms should be carefully considered even when PM2.5 is at low levels. C. elegans is a sensitive animal model for the evaluation of PM2.5 ecotoxicity.