Biomass burning emissions are a major contributor to ambient air pollution globally. After emissions, these air pollutants are subjected to several atmospheric chemical processes, such as photochemical oxidation and nitrate radical (night-time) aging. These processes can lead to the formation of new particles and alter the physicochemical characteristics of existing particles (secondary aerosols/changes). However, our understanding of how these processes affect in vitro cellular toxicity remains limited. Additionally, there have been no controlled human studies focused on air pollution exposures involving aged (secondary) air pollutants; most studies have used freshly emitted pollutants, which do not accurately reflect real-world air pollution composition. In this study, we exposed human lung epithelial cells (A549) to both freshly emitted and photochemically aged biomass burning air pollutants. We examined their effects on cytotoxicity, oxidative stress, and inflammation pathways. Our findings revealed that photochemical aging increases aerosol toxicity, leading to higher levels of oxidative stress and inflammation. Furthermore, we found that the toxicity was associated with chemical changes that occurred during aging, particularly in the O:C ratio.