The effects of mycorrhiza, Thiobacillus and Nitroxin (Azotobacter and Azospirillum sp.) biofertilizers under drought stress conditions with four levels of field capacity (FC) (control(100%), 85%, 70%, and 50%) on the antioxidant enzyme activities of medicinal pumpkin (Cucurbita pepo convar. pepo var. Styriaca) were evaluated during the years 2018–2019. Irrigation levels exhibited significant effects on all studied variables, except for the catalase (CAT) enzyme. A significant correlation was observed between the effects of irrigation levels and biofertilizers on antioxidant enzymes, soluble protein content, and grain yield. The highest activity of catalase and ascorbate peroxidase (APX) enzymes was achieved using mycorrhiza in 50% FC. Increasing drought intensity and mycorrhiza stimulated glutathione reductase (GR) and guaiacol peroxidase (GPX) activities by 32% and 66%, while Nitroxin increased them by 16% and 43%, respectively. Under severe drought stress conditions, only mycorrhiza exhibited a positive effect on GR and GPX enzymes. Under moderate and severe drought stress conditions, Nitroxin increased grain yield by 13% and 12.6%, respectively. The irrigation regimes and bio-fertilizers had a significant effect on β-sitosterol percentage. The highest amount was observed at the highest level of drought stress. Among the various bio-fertilizers treatments, the application of Thiobacillus yielded the highest percentage of β-sitosterol. The results of the present study demonstrate that the application of biofertilizers is beneficial in coping with drought stress.