•New methods for flotation agents combine microbiology and flotation theory.•Optimization and characterization of EPS produced by Azotobacter vinelandii.•Culture medium compositions affect the structure and composition of EPS.•Synthesized EPS for selective separation in chalcopyrite-chlorite flotation. In this study, we investigated microbial flotation as a green separation technology in mineral processing. We explored their potential use as environmentally friendly depressants for chlorite by extracting and purifying exopolysaccharides (EPS) from Azotobacter vinelandii (A.v) and comparing them with commercial alginate. Results showed that EPS derived from A.v under different fermentation substrates, particularly carbon sources, exhibited diverse structures, compositions and surface morphologies. Importantly, only some of these EPS’ variants demonstrated effective inhibition of chlorite. Scanning electron microscope (SEM) analysis revealed that the EPS with effective chlorite inhibition displayed a rough and porous surface morphology, resembling spherical-like structural units. We further investigated the different compositions of EPS using colorimetric methods, identifying total carbohydrates, uronic acids, acetyl and pyruvyl groups and proteins. In addition, we observed that functional groups, including acetyl group (CH3CO) and pyruvic acid (COCOOH), were detected in certain EPS variants, and these functional groups may be associated with the observed inhibition effect on chlorite. These findings highlight the potential of EPS derived from Azotobacter vinelandii as an environmentally friendly depressant for chlorite.