We show that arsenate can be readily reduced to arsenite on cell surfaces of common bacteria (E. coli or B. subtilis) or in aqueous dissolved extracellular polymeric substances (EPS) extracted from different microorganisms (E. coli, B. subtilis, P. chrysosporium, D. gigas, and a natural biofilm) in the absence of exogenous electron donors. The efficiency of arsenate reduction by E. coli after a 7-h incubation was only moderately reduced from 51.3% to 32.7% after knocking out the arsenic resistance genes (arsB and arsC). Most (>97%) of the reduced arsenite was present outside the bacterial cells, including for the E. coli blocked mutant lacking arsB and arsC. Thus, extracellular processes dominated arsenate reduction. Arsenate reduction was facilitated by removing EPS attached to E. coli or B. subtilis, which was attributed to enhanced access to reduced extracellular cytochromes. This highlights the role of EPS as a permeability barrier to arsenate reduction. Fourier-transform infrared (FTIR) combined with other chemical analyses implicated some low-molecular weight (<3 kDa) molecules as electron donors (reducing saccharides) and electron transfer mediators (quinones) in arsenate reduction by dissolved EPS alone. These results indicate that EPS act as both reducing agent and permeability barrier for access to reduced biomolecules in bacterial reduction of arsenate.