Aims: To determine the ability of a bismuth thiol to control floc formation in a multispecies population of micro-organisms obtained from the activated sludge unit of a wastewater treatment plant. The molecular level mechanisms by which bismuth-2-3-dimercapto-1-propanol (BisBAL) inhibits bioaggregation are also elucidated. Methods and Results: Micro-organisms were grown over a 3-day period in a batch system by adding glucose as an electron donor to stimulate short-term heterotrophic activity. Extracellular polymeric substances (EPS) produced by activated sludge micro-organisms during exponential and stationary growth phases in the presence and absence of BisBAL were characterized using colorimetry, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. BisBAL at its minimum inhibitory concentration (MIC, 10 mu mol l-1) was most effective in suppressing microbial floc formation. The principal effect of sub-inhibitory concentrations of BisBAL was to decrease total EPS production while largely preserving homology. Conclusions: Antifouling and bactericidal properties of BisBAL arise from its ability to reduce EPS expression and preferentially suppressing acidic and O-acetylated carbohydrates and certain protein secondary structures viz. beta-structures, random coils, and alpha-and 3-turn helices. As micro-organisms exhibited a much weaker tendency to aggregate at lower concentrations of these specific EPS components, they also appear to be important for the formation of microbial flocs and bioaggregates. Significance and Impact of the Study: BisBAL was shown to be highly effective against multispecies microbial aggregation. Novel bismuth-based biocides could also be potentially employed to control excess sludge production in wastewater treatment systems by inhibiting EPS expression.