Cucumber powdery mildew disease caused by Podosphaera xanthii (Castagne) U. Braun & Shishkoff severe disease-causing yield losses worldwide. This research study was conducted to evaluate the efficacy of the tested bio-agents, Trichoderma harzianum, T. viride, Bacillus subtilis, Paenibacillus polymyxa, and Serratia marcescens, as well as the fungicide score (Difenoconazole), on cucumber infected with P. xanthii, in vitro and under greenhouse conditions. Results indicated that culture filtrate of the tested bio-agents and the fungicide (control) significantly reduced P. xanthii conidial germination in vitro; the reduction percentage ranged between 91.17 and 76.06%. Also, score recorded the highest reduction percentage (97.19%). All treatments significantly decreased the disease severity and area under disease progress curve (AUDPC) post spraying the bio-agents on cucumber plants under greenhouse conditions. Score followed by B. subtilis significantly decreased disease severity percentage (67.33 and 65.38%, respectively) and AUDPC (322.84 and 342.06) than the untreated control (988.13 AUDPC). Additionally, treated cucumber plants showed a significant increase in plant growth parameters (plant height, total chlorophyll, fresh, and dry weight) and yield parameters (fruit number/plant and fruit weight/plant) as well the activity of defense-related enzymes, i.e., peroxidase (PO) and polyphenol oxidase (PPO), and total phenols content (TPC) compared to the untreated plants.