The oxidative potential (OP) of fine and coarse fractions of ambient aerosols was studied in the urban environment of Athens, Greece. OP was quantified using a dithiothreitol (DTT) assay, applied to the water soluble fraction of aerosol that was extracted from 361 fine and 84 coarse mode of 24-h and 12-h filter samples over a one-year period. During the cold period, samples were collected on a 12-h basis, to assess the impact of nighttime biomass burning emissions from domestic heating on OP. The chemical characteristics of aerosols were measured in parallel using an Aerosol Chemical Speciation Monitoring (ACSM) and a 7-wavelength Aethalometer. A source apportionment analysis on the ACSM data resulted in the identification of organic aerosol (OA) factors on a seasonal basis. A good correlation of OP with NO3−, NH4+, BC (Black Carbon), Organics and LV-OOA (low volatility oxygenated OA) was found during winter, revealing the importance of combustion and aging processes for OP. During the summertime, a good correlation between OP and SO4−2 and NH4+ indicates its association with regional aerosol – thus the importance of oxidative aging that reduces its association with any characteristic source. Multiple regression analysis during winter revealed that highly oxygenated secondary aerosol (LV-OOA) and, to a lesser extent, fresh biomass burning (BBOA) and fossil fuel (HOA) organic aerosol, are the prime contributors to the OP of fine aerosol, with extrinsic toxicities of 54 ± 22 pmol min−1 μg−1, 28 ± 7 and 17 ± 4 pmol min−1μg−1, respectively. In summer, OP cannot be attributed to any of the identified components and corresponds to a background aerosol value. In winter however, the regression model can reproduce satisfactorily the water soluble DTT activity of fine aerosol, providing a unique equation for the estimation of aerosol OP in an urban Mediterranean environment.