ABSTRACT The ozonation process of vegetable oils is commonly monitored by peroxide value (PV). However, PV methodology has been adapted from vegetable oils, with protocol divergences. In addition, few studies are based on the minimum level of ozonation to ensure biological activity. This study aimed to optimize the PV methodology for sunflower oil with different ozone dosage aiming to discriminate these samples. Also, to establish the relationship between its physicochemical properties with the biological activity. The optimized PV method was also applied to ozonated olive oil, as well as the other analytical techniques of iodine value (IV), acidity value (AV), density, viscosity, gas chromatography, infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), differential scanning calorimetry and thermogravimetry (DSC and TG). The PV method discriminated the ozonation level of the samples. Both ozonated sunflower and olive oils need to reach an intermediary ozone dosage to perform the in vitro bactericidal and fungicidal activity (MIC ≤2.5%), and anti-inflammatory activity (inhibition of nitric oxide release in Raw cells). These results point to the importance of standardizing the ozone dosage for each oil and the analytical methodologies used in its characterization.