Fine particulate matter (PM2.5) has been shown to cause oxidative stress, which has negative health consequences. The oxidative potential (OP) of PM2.5, a promising health exposure metric, was assessed in five Colombian cities using the synthetic respiratory tract lining fluid assay that tracks the depletions of glutathione and ascorbate. For this, a set of 91 integrated 2-week ambient PM2.5 samples were collected using Ultrasonic Personal Aerosol Samplers (UPAS) at background (5), traffic (37), industrial (12) and residential (37) sites. Across all site types, mean PM2.5 mass concentration was 20.20 ± 9.36 µg m− 3. The oxidative potential (OPAA for ascorbate and OPGSH for glutathione) varied widely across cities with an average of 2.67 ± 1.27 for AA and 2.93 ± 1.22 % depletion m− 3 for GSH. OP metrics among cities were not correlated with PM2.5 mass concentrations. Overall, industrial sites showed higher PM2.5 mass concentrations and OPAA. In contrast, OPGSH was not found to differ among industrial, traffic, or residential sites, but was lower for background sites. Our findings provide substantial evidence of variations in PM2.5 OP between cities and within the cities. Further research is needed to assess the association between OP and adverse health effects, as well as to attribute the sources that cause such variations.