Abstract: The bioactivity guided separation of the dichloromethane extract of the aerial parts of Macrotyloma uniflorum Linn. resulted in the isolation of methyl ester of hexadecanoic and ethyl ester of hexadecanoic acid mixture (I) and n-hexadecanoic acid (II). The structures of the isolated compounds were elucidated by spectroscopic analysis, including UV, IR, ¹H-NMR, ¹³C-NMR and mass spectroscopy. In addition, the fractionated crude extract of 1-butanol exhibited the significant hemolytic activity by using mouse erythrocytes.