This research presents the first study findings on the phytochemical contents and anticancer and antioxidant activities of galls caused by Eriophyidae mites on the leaves of Juglans regia L. Gall extracts collected from different localities in Turkey and prepared with solvents of different polarity were investigated for both antioxidant and cytotoxic activity. Cytotoxic activity studies showed that MCF-7 cancer cells were more sensitive to WLAA extract at a concentration of 100 μg/mL compared to healthy HUVEC cell lines. LC-QTOF-MS analysis results showed that all extracts contain chlorogenic acid, quercetin 4'-O-glucoside/quercetin 3-O-galactoside, quercetin 7-xyloside/quercetin 3-O-arabinoside, quercetin 7-O-rhamnoside, kaempferol 3-O-xyloside/kaempferol 3-O-arabinoside, and kaempferol derivatives. It was concluded that polyphenolic extracts obtained from galls formed in J. regia leaves can be considered as a new potential natural source for drug development studies due to their antioxidant and cytotoxic effects.