The impact of inhalable fine particulate matter (PM2.5, aerodynamic diameter <2.5 μm) on public health is of great concern worldwide. Knowledge on their harmful effects are mainly due to studies carried out with whole air particles, with the contribution of their different fractions remaining largely unknown. Herein, a set of urban PM2.5 samples were collected during daytime and nighttime periods in autumn and spring, aiming to address the seasonal and day-night variability of water-soluble organic matter (WSOM) composition. In vitro analysis of the oxidative and pro-inflammatory potential of WSOM samples was carried out in both acute (24 h) and chronic (3 weeks) exposure setups using Raw264.7 macrophages as cell model. Findings revealed that the structural composition of WSOM samples differs between seasons and in a day-night cycle. Cell exposure resulted in an increase in the transcription of the cytoprotective Hmox1 and pro-inflammatory genes Il1b and Nos2, leading to a moderate pro-inflammatory status. These macrophages showed an impaired capacity to subsequently respond to a strong pro-inflammatory stimulus such as bacterial lipopolysaccharide, which may implicate a compromised capacity to manage harmful pathogens. Further investigation on aerosol WSOM could help to constrain the mechanisms of WSOM-induced respiratory diseases and contribute to PM2.5 regulations.