Abstract Flocculation as a pre-separation method can help make production of biodiesel from microalgae economically feasible. In a previous study, Moringa oleifera seed flour (1 g.L-1) was shown to be a very efficient flocculant for Chlorella vulgaris, a microalga with high potential for biodiesel production. In this study, several aspects of C vulgaris flocculation mediated by Moringa were investigated in order to optimize the separation of this biomass. Flocculation efficiency was the same with seeds from different origins and lots. The stationary growth stage was best for harvesting C vulgaris cells to carry out flocculation efficiently (93%). The use of flour extracts and cake extracts generated the best cost-benefit ratio (flocculation efficiency from 78 to 97% with a saving in mass of seed of 75%). The highest efficiency was reached with extracts prepared with seawater and NaCl solutions which have high salt concentration. Reasonable stability of the extract allows its use for up to two weeks, provided it is kept at low temperature (4 ºC).