Olive oil mill wastewaters (OMWs) show significant polluting properties due to their content of organic substances, and because of their high toxicity toward several biological systems. Wastewaters' toxicity has been attributed to their phenolic constituents. A chemical study of wastewaters from a Ligurian oil mill characterized phenolic products such as 1,2-dihydroxybenzene (catechol), derivatives of benzoic acid, phenylacetic acid, phenylethanol, and cinnamic acid. The OMWs were fractioned by ultrafiltration and reverse osmosis techniques and tested for toxicity on aquatic organisms from different trophic levels: the alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum); the rotifer Brachionus calyciflorus; and two crustaceans, the cladoceran Daphnia magna and the anostracan Thamnocephalus platyurus. The fraction most toxic to the test organisms was that from reverse osmosis containing compounds of low molecular weight (<350 Da), and this was especially due to the presence of catechol and hydroxytyrosol, the most abundant components of the fraction.