Flocculation is a promising approach for reducing the cost of harvesting microalgae. Flocculation of microalgae can be induced by precipitation of calcium phosphate (Ca-phosphate) when pH increases above 8.5, a pH level that can be achieved by simple photosynthetic CO2 depletion. Using the freshwater microalgae Chlorella vulgaris as a model, we identified the combinations of minimum pH and Ca and PO4 concentrations to induce flocculation. Predicted concentrations of amorphous Ca3(PO4)2 precipitation (chemical modelling) explained flocculation in these solutions. The efficiency of flocculation decreased with increasing microalgal biomass concentration. Solution renewal experiments suggest that flocculation is inhibited by algal organic matter in the medium, even when present at relatively low concentrations relative to concentrations in stationary phase medium. Addition of dissolved organic compounds showed that organic acids with a high molecular weight (e.g. humic acids, alginate) have a strong inhibitory effect on flocculation whereas glucose or acetate had no such effect. These effects may be related to complexation of Ca2+ or effects of organic matter on growth of the Ca-phosphate crystals. Precipitation of Ca-phosphate in media with high organic matter content requires a high water hardness (500 μmol L−1 Ca) and high PO4 concentrations (350 μmol L−1 P). Flocculation can be facilitated by addition of surplus PO4 to the medium. This surplus PO4 may be recovered after flocculation by re-dissolution of the Ca-phosphate through mild acidification.