Numerous polyphenolic compounds originating from plants are thought to have anticancer and apoptosis-inducing effects on cancer cells. Plumeria rubra (family Apocyanaceae) is utilized as traditional medicine to cure a variety of diseases. It is well known for being a decorative tree, broad cultivated, very fragrant, and having a wide range of secondary metabolites, which inspired us to learn more about its possible medical uses. In this work, the total phenolic content of the methanol extract of P. rubra was established spectrophotometrically and found to be 11.05 mg/g, while the total flavonoid content was found to be 29.22 mg/g. Fifteen polyphenolic compounds were identified by the HPLC analysis of P. rubra methanol leaves extract. Additionally, an in vitro cell viability test was used to assess the cytotoxic activity of the methanol leaves extract of P. rubra. The findings of the cytotoxicity test revealed substantial anticancer activity against the human lung carcinoma A-549 cell line with an IC50 of 41.72 μg/ml and less activity against the human colon carcinoma HCT-116 and the human breast carcinoma MCF-7 cell line with IC50s of 57.62 and 75.8 μg/ml, respectively. Results of this work demonstrate that methanol leaves extract of P. rubra is a valuable source of phenolics and flavonoids that that can substantially slow the spread of cancer.