Eight new mono-tetrahydrofuran (THF)-type annonaceous acetogenins, squafosacins B, C, F, and G (1-4), squadiolins A-C (5-7), and cis-annotemoyin-1 (8), as well as eight known annonaceous acetogenins, glabranin, annotemoyins-1 and -2, bullatencin, cis-bullatencin, and uvariamicins-I, -II, and -III, were isolated from the seeds of Annona squamosa by HPLC. The structures of all new isolates were elucidated by using spectroscopic and chemical methods. Squadiolins A (5) and B (6) showed ng/mL potency against human Hep G2 hepatoma cells and significant cytotoxic activity against human MDA-MB-231 breast cancer cells. Squafosacin B (1) also exhibited significant cytotoxic activity against human Hep G2 and 3B hepatoma and MCF-7 breast cancer cells. In addition, the chelation of mono-THF acetogenins with calcium ions was investigated using isothermal titration calorimetry.