Root exudates are known to influence the rhizosphere environment, yet their potential as sources of anticancer compounds remains underexplored. This study investigated the chemical composition and anticancer activity of root exudates from hydroponically cultivated tomato (Solanum lycopersicum), cucumber (Cucumis sativus), lettuce (Lactuca sativa), and spinach (Spinacia oleracea). Root exudates were collected under sterile conditions and analyzed for primary (carbohydrates, amino acids, organic acids) and secondary metabolites (phenolics, flavonoids, tannins, alkaloids, steroids, and saponins). Secondary metabolites varied significantly among the different plants, with tomato exhibiting the highest levels, while primary metabolites showed no significant differences. Cytotoxicity was evaluated against six human cancer cell lines (CLS-145, AsPC-1, HCT116, HepG2, KYSE-410, MCF-7) using the MTT assay. Tomato root exudates exhibited potent anticancer activity, with IC50 values 64–75 % lower than doxorubicin, while exudates from other species displayed moderate effects. High-content screening assay further demonstrated that tomato exudates induced increased plasma membrane permeability, nuclear condensation, mitochondrial membrane depolarization, and elevated cytochrome c release, indicating apoptosis via the intrinsic pathway. qPCR analysis confirmed significant upregulation of pro-apoptotic genes (BAX, CASPASE-3) and cell cycle regulators (p21), alongside downregulation of anti-apoptotic (BCL2) and proliferation-associated genes (CDK1, Cyclin B1). These findings highlight the rich secondary metabolite profile and strong anticancer potential of tomato root exudates, suggesting their promising application in plant-based cancer therapeutics and expanding the functional significance of root exudation in the rhizosphere.