Ultraviolet (UV) light has exhibited antimicrobial effects, with recent studies looking at UV-A light in particular. The objective of this study was to determine the antimicrobial mechanism and microbial inactivation kinetics of UV-A light on processed cheese. Processed cheese was inoculated with Escherichia coli K12 and Listeria innocua to yield a final concentration of ∼5 log[colony-forming units (CFU)/g] and then exposed to UV-A light for 0–60 min to determine the kinetics of microbial inactivation. The experimental data were fitted with the Weibull model of inactivation kinetics. To achieve an ∼6 log(CFU/g) decrease, UV-A light exposure for ∼70 min was required for E. coli K12 and ∼130 min for L. innocua L2. The processed cheese was analyzed using infrared spectroscopy after UV-A exposure for 0 and 60 min and showed no apparent changes in the surface chemistry. A decrease in the moisture content was noted, which caused an increase in the concentration of lipids on the surface. A statistically significant (P < 0.05) effect was observed in the color of the cheese after UV-A light exposure for 60 min. The effect of UV-A light exposure on the oxidative stress and membrane damage of both bacteria was analyzed through fluorometric techniques. A significant (P < 0.05) increase in oxidative stress and membrane damage was observed for both bacteria, which was more pronounced for E. coli K12. Our findings suggest the UV-A light could prove to be a suitable alternative for surface decontamination of dairy products.