This study investigates the cytotoxic effects of R. tridentata extracts on prostate cancer cells, providing insight into its potential therapeutic benefits and scientific validation for its traditional use in cancer treatment. The cytotoxicity of R. tridentata extracts was evaluated on prostate cancer cell lines (LNCaP and DU 145) using the MTT assay, with doxorubicin as a reference standard. Our findings demonstrated significant concentration- and time-dependent cytotoxic effects of the extracts on both cell lines (p < 0.0024 to p < 0.0002). Notably, the methanol extract exhibited potent cytotoxicity, with IC values ranging from 124.07 to 211 μg/mL (DU145) and 100 to 180 μg/ml (LNCaP) over 24-72 h (p < 0.0024 to p < 0.0001), highlighting its therapeutic potential. This study unveils the promising cytotoxic activity of R. tridentata extracts, particularly methanol extracts, against prostate cancer cells, showcasing their concentration- and time-dependent effects. These findings underscore the potential of plant-derived extracts as therapeutic agents for cancer treatment, warranting further research.