Ozonation reactions are very important in fatty acid chemistry since their ozonation products are involved in vital biological processes. When an unsaturated acid reacts with ozone in presence of an organic solvent, Criegee ozonides are produced according to the Criegee mechanism. These ozonides are relatively stable compounds, thus, in the organism they have biological effects at distant sites from the site they are formed. The antimicrobial activity of ozonized sunflower oil (O1eozonR) has been previously well documented. We have suggested that ozonides are involved in the antimicrobial effect of O1eozonR/. In this study we try to demonstrate this hypothesis. Ozonides were obtained by total reaction of ozone gas with a sample of methyl oleate in an organic solvent (hexane). Proton Nuclear Magnetic Resonance (H NMR) technique was used for identification of ozonides in ozonation products, whereas biological evaluation was done by determining Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) of ozonation products of different microorganism. The ozonide signal was identified in the O1eozonR and ozonized methyl oleate while in sunflower oil was not. Ozonation products from methyl oleate, in our reaction conditions, were spectroscopically found to be only ozonides. MICs for ozonides from ozonized methyl oleate were from 2.3 to 28 mg/mL, whereas MBC and MFC were 460 mg/mL comparable with those from O1eozonR. These results demonstrate that ozonides are one of the active species of O1eozonR.