The increasing resistance of pathogens to conventional antibiotics has necessitated the search for novel antimicrobial agents from medicinal plants. Nelsonia canescens, a plant traditionally used in African and Asian in the management of diseases such as viral infections, cardiovascular, and inflammation, have been reported with antimicrobial activity, was investigated for its bioactive constituents. The whole plant was collected, air-dried, and extracted using 70% methanol. The crude methanol extract was partitioned into hexane, chloroform, ethyl acetate, and butanol fractions respectively. The ethyl acetate fraction was subjected to column chromatography and gel filtration, leading to the isolation of a compound coded A₁. The structure of compound A₁ was established through UV, FTIR, NMR (¹H, ¹³C, DEPT, COSY, HMQC, HMBC), and chemical tests. Compound A₁ was identified as 2', 4'-dihydroxy-3-ethanol-5-(1→4) glucose-rhamnose chalcone, a flavonoid derivative. Spectral analysis confirmed its structure, with key signals including olefinic protons (δ 6.30 and 7.62) in the trans configuration, aromatic protons, and sugar moieties. The compound exhibited a melting point of 105–107°C and was sparingly soluble in chloroform but fully soluble in methanol suggesting that the compound is highly polar in nature. This is the first report of isolation of 2', 4’-dihydroxy-3-ethanol-5-(1→4) glucose-rhamnose chalcone from Nelsonia canescens, and new to the genus contributing to the taxonomy of the plant. The compound’s structural features suggest potential bioactive properties, warranting further investigation into its pharmacological applications through in vitro and molecular docking studies.