Walnuts are one of the healthiest foods in the world because they are one of the best sources of beneficial nutrients, minerals, and antioxidants. They also contain key sources of energy. Despite several traditional uses, the leaves of Juglans regia L. have received little attention regarding phytochemical and pharmacological potential. Thus, the current study intended to find the walnut genotypes with the greatest antioxidant, anticancer, and antifungal activity. The total polyphenolic, flavonoid, and flavanol contents of leaves from 14 walnut genotypes were determined. Genotypes that accumulate flavonoid/flavonol contents (99.8–111.93 mg/g quercetin equivalent (QE) and 101.67–111.83 mg/g QE) showed significantly higher ferric reducing antioxidant potential (FRAP) activity (128.2–148.1 μM Fe²⁺/g dry weight (DW)] than other genotypes. Maximum divergence in the quercetin content (0.8–1.23 mg/g) of walnut genotypes was obtained by cluster analysis. The active component, quercetin, was measured using RP-HPLC. Moreover, the extracts were investigated for antifungal and anticancer assays. We report the significant antifungal potential of walnut leaf genotypes against Candida glabrata, Candida albicans, and Candida tropicalis, with 57.7–93.6%, 26.8–51.5%, and 26.8–51.5% inhibition, respectively. The most significant antiproliferative effect was shown by Opex Culchry, which exhibited 9.4% cell viability at a concentration of 25 µL (0.75 mg) against lung (A549) cell lines. Chenovo exhibited 2.9, 6.2, and 2.2% cell viability, Opex Culchry exhibited 2, 1.5, and 2.4% cell viability, and Sulieman showed 7.6, 0.9, and 7% cell viability against the colon (HCT116) cell lines. The results showed that walnut leaves possess enormous potential as antioxidants, and as anticancer and antifungal agents.