Two diastereomeric 4-methylene-2-cyclohexenones, otteliones A and B (1A and 1B), have been isolated from the freshwater plant Ottelia alismoides collected in the Nile Delta, Egypt. These natural products show remarkable in vitro cytotoxicity against various cancer cell lines. Ottelione B proved to have a constitution identical to that of the known (constitution only) ottelione A. The relative configurations of both 1A and 1B were studied by NOE and by the combination of molecular modeling and 1H NMR coupling constant analysis. A unique relative configurational assignment (1α,3β,3aβ,7aα) was deduced for 1B; 1A was assigned one of two alternative diastereomers (1α,3α,3aα,7aα or 1α,3α,3aβ,7aβ).