Background The industry of olive oil produces large wastewater quantities. The elimination of these toxic by-products is an important environmental issue that requires a sufficient solution. In this study, Scenedesmus sp. was used for the simultaneous lipid and antioxidant molecules production and olive mill wastewater biodegradation.Results The two-stage olive mill wastewater cultivation did not significantly inhibit the production of microalgal biomass, compared to a single growth stage, when OMW and OMUF were supplied at the beginning of cultivation. OMUF, at 40%, enhanced the Ch a, b and carotenoids amounts (2.57, 7.4 and 1.69 mg/g dw, respectively), and protein and carbohydrate accumulation (350.96 and 315.62 ng/cell, respectively), after single phase culture. However, the highest OMUF and OMW concentrations increased total lipid content to 639. 13 and 679.86 ng/cell, respectively. About 58% of phenol removal was achieved and highest accumulation of polyphenols in algal cells with 40% OMUF condition under one stage strategy. The highest OMUF and OMW concentrations exhibited highest DPPH and ABTS scavenging ability after single and two-phase culture. Moreover, the cytotoxicity of the OMUF against HEK- 293 human embryonic kidney cells was evaluated by MTT assay. The MTT assay showed that the biomass of Scenedesmus sp. cultured on Tunisian filtered olive mill waste water did not shown any toxic effect.Conclusion Our results indicate that Scenedesmus sp. is a promising microalga for the biotreatment of OMW and the production of bioactive metabolites.