The purpose of this investigation was to compare the effectiveness of the tested biological control treatments, i.e., Trichoderma album, T. harzianum, T. hamatum and T. viride; biopesticides, such as Blight Stop (T. harzianum), which is used at the rate of 1 L/100 liters of water; and additionally natural compounds, such as micronized sulfur fungicide, which is utilized at a rate of 250 gm/100 liters of water in decreasing germination and severity of Podosphaera fuliginea powdery mildew disease on cucumber cv. Hybrid 20 to the control under in vitro and greenhouses. The treatment with Micronic sulfur fungicide reduced inhibitory germination percentages and disease severity the most (92.69 and 87.30%), followed by the T. harzianum isolate (86.96 and 81.71%) as compared to the control treatment. The application of cucumber plants with T. harzianum at a rate of 1:100 exhibited the greatest decrease in the inhibition of disease severity and the greatest increase in yield, biochemical analysis, including total chlorophyll, total phenolic content, peroxidase (PO), and polyphenol oxidase (PPO) enzyme activities throughout the growing season 2024 under greenhouse condition, compared to untreated plants. On the other hand, T. hamatum demonstrated the least response from treatments. There was no observable, major variations between the other treatments and the control.