<p>The <i>Euphorbiaceae</i> family <i>Euphorbia pulcherrima</i> is well known for its anticancer properties. The research examines the roles of two flavonoids found in <i>E. pulcherrima</i> in the inhibition of thymidine phosphorylase (TP), an enzyme in cancer development, metastasis, and chemotherapy resistance. This study was designed to evaluate the in vitro TP inhibitory activity of two flavonoids isolated from <i>E. pulcherrima</i> and to investigate their potential binding modes and interactions with TP using molecular docking analysis. In the current studies, the chemical constituents of <i>E. pulcherrima</i> were isolated and characterized. Both of the constituents were flavonoids, namely—5,7,8,3′,4′-pentahydroxy-3-methoxyflavone (Flavonoid <b>1</b>) and kaempferol-3-β-D-glucopyranosyl (Flavonoid <b>2</b>). Both of the flavonoids were evaluated spectrophotometrically for TP inhibitory activity as compared to the 7-deazaxanthine, and the IC₅₀ values were determined. Molecular docking was performed to explore the protein–ligand interactions at the TP active site. Both the flavonoids significantly antagonized TP. The maximum inhibitory effect of flavonoid 1 was 83.60% at 0.2 µM and an IC₅₀ of 12.60 ± 1.00µM. At a concentration of 0.2 µM, flavonoid 2 showed 78.09% TP inhibition, with an IC₅₀ of 19.09 ± 1.40 µM. These findings were supported by docking results according to which Flavonoid 1 had a better predicted binding affinity (−8.5 kcal/mol) than Flavonoid 2 (–4.8 kcal/mol). Moreover, Flavonoid 1 was predicted to exhibit better drug-like properties and increased bioavailability compared to Flavonoid 2, whose sizeable sugar group reduced the compound’s predicted bioavailability. The results indicate Flavonoid <b>1</b> is a promising anti-cancer lead compound, as it has a strong TP inhibition, good pharmacokinetic profiles, and low toxicity. Further preclinical testing of Flavonoid <b>1</b> should be done.</p>