A new ellagic acid derivative, 3-O-methyl ellagic acid dihydrate ( 4 ), along with three known phytoconstituents, 4-acetylresorcinol ( 1 ), 4-hydroxyphenylmethyl ketone ( 2 ), and docosanoic acid docosyl ester ( 3 ), were isolated from the leaves of Bixa orellana L. for the first time. Their structures were elucidated on the basis of chemical and physical data (IR, UV, 1 H NMR, 13 C NMR, and mass). Herein, we report the extraction, isolation, characterization, and biological activities of phytoconstituents isolated from the leaves of Bixa orellana. The structure of compound 4 was further confirmed by X-ray crystallographic studies. The isolated compounds 1–4 were screened for their antimicrobial activity by the disc diffusion method using ciprofloxacin and amphotericin B as standard drugs. Moreover, compound 4 was evaluated for its plausible anticancer activity using MTT assay.