Biological flocculants are advantageous due to their biodegradability and safety to living beings. In this work, four bacterial strains, isolated from sugarcane cropped soil (Bacillus megaterium LBPMA-APFSG3Isox and B. toyonensis LBPMA-ACOPR1.Isox) and sludge of an agroindustrial effluent (B. pumilus LBPMA-BLD07 and B. thuringiensis LBPMA-EFIII), were studied. It was found that all of them secreted bioflocculants, at 37 ± 1 °C, with no pH changes over time and the flocculant activity increased during the time course of incubation. These results stimulated the optimization of the culture conditions to improve flocculation rates, such as the pH, nitrogen sources and carbon. For B. toyonensis LBPMA-ACOPR1.Isox and B. thuringiensis LBPMA-EFIII, the best pH for the bioflocculant production was 5.0. Sucrose and maltose were the best sources of carbon, while urea was the preferred source of nitrogen for two of the tested isolates (B. pumilus LBPMA-BLD07 and B. toyonensis LBPMA-ACOPR1.Isox), followed by (NH 4)2SO 4 (B. megaterium LBPMA-APFSG3Isox) and peptone (B. thuringiensis LBPMA-EFIII). The FTIR-ATR spectra of each extracted material responsible for the flocculant activities of the strains displayed carboxyl, hydroxyl and methoxy functional groups characteristic of polysaccharides.