Microbial extracellular polymeric substances (EPS) were produced in two membrane bioreactors, each separately treating fresh and saline synthetic wastewater (consisting of glycerol and ethanol), with the purpose of applying them as sustainable bioflocculants. The reactors were operated under nitrogen-rich (COD/N ratios of 5 and 20) and limited (COD/N ratios of 60 and 100) conditions. Under both conditions, high COD removal efficiencies of 87-96% were achieved. However, nitrogen limitation enhanced EPS production, particularly the polysaccharide fraction. The maximum EPS recovery (g EPS - COD/g CODinfluent) from the fresh wastewater was 54% and 36% recovery was obtained from the saline (30 g NaCl/L) wastewater. The biopolymers had molecular weights up to 2.1 MDa and anionic charge densities of 2.3-4.7 meq/g at pH 7. Using kaolin clay suspensions, high flocculation efficiencies of 85-92% turbidity removal were achieved at EPS dosages below 0.5 mg/g clay. Interestingly, EPS produced under saline conditions proved to be better flocculants in a saline environment than the corresponding freshwater EPS in the same environment. The results demonstrate the potential of glycerol/ethanol-rich wastewater, namely biodiesel/ethanol industrial wastewater, as suitable substrates to produce EPS as effective bioflocculants.