Abstract The bio-flocculants used in this study were synthesised by the Mannich reaction, which includes three reagents: a substrate (tannin extracts of Acacia, Quebracho, and Castanea), formaldehyde, and an amine derivative (ethanolamine, diethanolamine, ammonium chloride). Nine natural flocculants were prepared by combining extracts and amines; these products were evaluated in three different wastewater samples in two experimental phases. In phase I, five physicochemical parameters were analysed. From the data obtained, a multivariate, completely randomised design (CRD-Manava) was used, with a factorial arrangement and mean plots. In phase II, the three bio-flocculants with the most statistically significant responses and their mixtures were examined, evaluating 14 biological and physicochemical parameters. Statistical analysis was guided in this phase by CRD blocks, finding a significant removal in the physicochemical parameters analysed in the different types of wastewater and obtaining removal rates between 50 and 90%, depending on the parameter. At the end of both phases, the bio-flocculants acacia-ammonium chloride and quebracho-diethanolamine were the most efficient in the removal of turbidity (34–99%), true colour (93–100%) and total solids (12–99%). In addition, the natural flocculants showed low mutagenicity index (MI: 0.33–0.93) compared to aluminium sulphate (MI: 4.87–8.81).