Positively charged gold nanoparticles [(+) AuNPs] of 5-20 nm were synthesized by using electrochemically active biofilm (EAB) formed on a stainless steel mesh, within 30 minutes, in aqueous solution containing HAuCl4 as a precursor and sodium acetate as an electron donor. Electrochemically active bacteria present on biofilm oxidize the sodium acetate by producing electrons. Simultaneously, stainless steel also provides electrons because of the Cl- ions penetration into the stainless steel. Combined effect of both the EAB and stainless steel mesh enhances the availability of electrons for the reduction of Au3+ in the solution, which makes this synthesis efficient and fast. Therefore, small size, positively charged (+32.72 mV), monodispersed, controlled, easy separation and extracellular synthesis of (+) AuNPs makes this protocol highly significant. As-synthesized AuNPs were characterized by UV-vis, DLS, XRD, TEM, HRTEM, EDX and SAED. (+) AuNPs shows remarkable enhancement in the rate of reduction of methyl orange by NaBH4 because of the electron relay effect.