Cucurbits plants are very susceptible to fungal diseases as powdery mildew (PM) infection. Currently, the PM early detection in the field is the cutting-edge research. The objectives of our study were to assess visible and near-infrared spectroscopy of normal and infected leaves for early detection of PM in Cucurbita pepo L. plants. Samples for spectral analysis were taken three days a week (Monday, Wednesday, and Friday) after the first true leaves appeared. The reflectance spectra of leaves were collected using an HR4000CG-UV-NIR spectrometer (Ocean Optics) with a fiber optics probe. Vegetative indices were used for discrimination between infected and healthy plants. The calculated vegetation indices (green normalized difference vegetation index, triangular greenness index, single-band reflectance index, simple ratio indices, and anthocyanins reflectance index) showed the highest sensitivity to differentiate healthy, infected plants at a symptomless stage, first symptoms, and diseased plants. The best prediction on early detection in sampling days were structure-independent pigment index and red-edge reflection point with an R of 0.211 and 0.1893, respectively. Our study shows the efficacy of the identification based on reflectance spectra for an early distinction of PM disease and could be used in cucurbits plants with similar characteristics on leaves.