Background: Stigmasterol is an unsaturated phytosterol that belong to the class of tetracyclic steroids abundant in Rhoicissus tridentata. Stigmasterol is an important constituent since it has shown impressive pharmacological effects such as anti-osteoarthritis, anticancer, anti-diabetic, anti-inflammatory, antiparasitic, immunomodulatory, antifungal, antioxidant, antibacterial, and neuroprotective activities. Furthermore, due to the presence of π system and hydroxyl group, stigmasterol is readily derivatized through substitution and addition reactions, allowing for the synthesis of a wide variety of stigmasterol derivatives. Methods: Stigmasterol (1) isolated from Rhoicissus tridentata was used as starting material to yield eight bio-active derivatives (2–9) through acetylation, epoxidation, epoxide ring opening, oxidation, and dihydroxylation reactions. The structures of all the compounds were established using spectroscopic techniques, NMR, IR, MS, and melting points. The synthesized stigmasterol derivatives were screened for cytotoxicity against the hormone receptor-positive breast cancer (MCF-7), triple-negative breast cancer (HCC70), and non-tumorigenic mammary epithelial (MCF-12 A) cell lines using the resazurin assay. Results: Eight stigmasterol derivatives were successfully synthesized namely; Stigmasterol acetate (2), Stigmasta-5,22-dien-3,7-dione (3), 5,6-Epoxystigmast-22-en-3β-ol (4), 5,6-Epoxystigmasta-3β,22,23-triol (5), Stigmastane-3β,5,6,22,23-pentol (6), Stigmasta-5-en-3,7-dion-22,23-diol (7), Stigmasta-3,7-dion-5,6,22,23-ol (8) and Stigmast-5-ene-3β,22,23-triol (9). This is the first report of Stigmasta-5-en-3,7-dion-22,23-diol (7) and Stigmasta-3,7-dion-5,6,22,23-ol (8). The synthesized stigmasterol analogues showed improved cytotoxic activity overall compared to the stigmasterol (1), which was not toxic to the three cell lines tested (EC50 ˃ 250 µM). In particular, 5,6-Epoxystigmast-22-en-3β-ol (4) and stigmast-5-ene-3β,22,23-triol (9) displayed improved cytotoxicity and selectivity against MCF-7 breast cancer cells (EC50 values of 21.92 and 22.94 µM, respectively), while stigmastane-3β,5,6,22,23-pentol (6) showed improved cytotoxic activity against the HCC70 cell line (EC50: 16.82 µM). Conclusion: Natural products from Rhoicissus tridentata and their derivatives exhibit a wide range of pharmacological activities, including anticancer activity. The results obtained from this study indicate that molecular modification of stigmasterol functional groups can generate structural analogues with improved anticancer activity. Stigmasterol derivatives have potential as candidates for novel anticancer drugs.