Pumpkin is a widely recognized plant in the Cucurbitaceae family because of its significance as a pharmaceutical food. Ozonized oils are becoming more popular among researchers and being employed in medical facilities due the continuous search for powerful biomedical medicines that can combat illnesses with controllable negative repercussions. Pumpkin seed oil was ozonized at an average rate of 0 to 8 L/minute. The chemical composition of the oil before and after treatment was characterized using Gas Chromatography-Mass Spectrometry (GC-MS) analysis. Anti- Helicobacter pylori (H. pylori) of both unrefined and ozonized pumpkin oils were detected using agar well diffusion technique. The anti-biofilm was tested at 25, 50 and 75% of MBC. Time killing assay was done to compare the antimicrobial impacts both refined and ozonized oils the various of the antibacterial impact at various time points. Anticancer role versus human prostate cancer cell line (PC-3) cells for oil forms was done using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT protocol. The change of cell cycles was compared in cancer cells and after treatment by examined oil forms. In vitro alpha glucosidase, alpha amylase and lipase enzymes inhibition was done in both oil samples relative to norms. Both oil forms contained 25 bioactive molecules where ozone exposure altered the molecular diversity with increased the number of classes of molecules in ozonized oil. Additionally, exposure to ozone significantly increased the levels of and n-propyl 9,12-octadecadienoate and 9-Hexadecenoic acid, 9-hexadecenyl ester, (Z, Z)- could be seen with high level in ozonized oil. A slight rise in anti-H. pylori could be seen in ozonized oil which showed bactericidal impact which had been confirmed by time killing assay. The anti-biofilm tested activity reached its maximal level upon using 75% of MBC of ozonized pumpkin seed oil. The ozonized oil had a promising anticancer role towards PC-3 cells through induction of early apoptosis. Exposure of pumpkin oil to the ozone led to improvement in in vitro antidiabetic and anti-obesity roles. These findings support the conclusion that being exposed to pumpkin oil to ozone improves the oil's in vitro impact to pathogenic microbes and diseases that are not transmissible to be applied in preclinical applications in future studies.