Fatty acid derivatives, (Z)-2-hydroxy-3-(octadec-9-enoyloxy) propanoic acid (1) and Hexadecanoic acid-2,3- dihydroxy propyl ester (2) were isolated from the seeds of Annona squamosa. Compound 1 is novel and isolated for the first time from a plant source and 2 is reported for the first time from A. squamosa. Compounds 1 & 2 were evaluated for cytotoxic activity in vitro against human cancer cell lines (A549, ACHN, HELA, PC-3, B-16, HT-29, and MCF-7). Compound 1 showed potential cytotoxicity against HELA (IC50 5.45μg/ml) and MCF-7 (IC50 4.66μg/ml) cell lines where as 2 showed potential cytotoxicity against HELA (IC50 9.16μg/ml) cell lines.