The present study was aimed to evaluate the cytotoxic potential of hydroalcoholic extract of Annona squamosa L. using Allium cepa test and Brine Shrimp Lethality assay. Fresh and ripe sugar apple fruits were collected from an orchard at Ota-Efun, Olorunda Local Government, Osogbo, Nigeria (07^o  32¢ 30.2496² N, 04^o 31¢ 41.7036² E) and their identity authenticated at IFE Herbarium, Department of Botany, Obafemi Awolowo, University, Ile-Ife, Nigeria. The seeds were collected, fermented locally for 7days, and oven-dried. The cotyledon was separated from the seed coat, pulverized, defatted, and exhaustively extracted with 80% ethanol. The extract was concentrated and air-dried and named Annona squamosa Fermented Cotyledon Hydroalcoholic Extract (AFCHE). The extract was screened for phytoconstituents on GC-MS machine. Allium cepa test and brine shrimp lethality bioassay were used to screen for the cytotoxic potential of AFCHE. The root length and protein concentration of the onion root after 7 days of exposure showed a significant decrease with an increase in AFCHE concentration and an increase in percentage growth inhibition of the root. The LC₅₀ obtained from the brine shrimp lethality bioassay qualified AFCHE as a highly toxic substance and a good anticancer agent worth screening.