<p>Cucumber is important vegetable but downy mildew caused by <i>Pseudoperonospora cubensis</i> reduces its yield and affected its production worldwide. In the present study, two cucumber varieties (Kheera No.1 and Punjab Naveen) were artificially inoculated at three leaf stage with <i>P. cubensis</i> to assess the physiological, biochemical and anatomical changes in cucumber plants. Kheera No.1 was found to be moderately resistant while Punjab Naveen as moderately susceptible according to disease severity index. The result revealed that the total chlorophyll content and electrolyte leakage decreased more in inoculated plants of the variety Punjab Naveen from 2 to 12 DAI. Post infection, a decrease in total soluble sugar content was recorded in both the varieties. However, an increase in total soluble proteins, total phenols, total flavonoids, ascorbic acid were exhibited with more enhancement in Kheera No.1 in contrast to Punjab Naveen. The activities of various antioxidant enzymes were found to increase in inoculated plants of both the varieties as compared to their respective controls. Phylloplane morphology and leaf anatomical studies at 8 DAI (Days after inoculation) showed structural variations between the healthy and infected leaves of both the varieties. The size of stomata, stomatal index, total leaf thickness, spongy and palisade tissues were found to be higher in Punjab Naveen, but trichome size and frequency were higher in Kheera No. 1. Therefore, favourable phylloplane morphology and leaf anatomy, enhanced antioxidants activity, total soluble proteins and decline in the level of total soluble sugars, post infection, helped in imparting moderate resistance to the pathogen infection in variety Kheera No.1.These indices can be used to screen large population of cucumber for tolerance to the disease.</p>