An electrochemically active biofilm (EAB) was used for the synthesis of positively charged gold nanoparticles [(+)AuNPs] and in-situ hydrogen production without any external energy input. The EAB generate electrons and protons by decomposing sodium acetate (carbon source) in water at 30 C. These electrons were used initially to reduce Au3+ to Au0, and later in-situ, these generated electrons and protons were used for hydrogen production. The as-synthesized (þ)AuNPs acted as catalyst by providing a charged surface to reduce the protons, leading to the formation of molecular hydrogen according to the Volmer-Heyrovsky mechanism. The hydrogen produced was confirmed and estimated by gas chromatography and a fuel cell test, respectively. The maximum rate of hydrogen production reached ~105 � 2 mL/L day. This suggests that hydrogen production is possible in a single chamber reactor using an EAB in the presence of sodium acetate as a substrate and (+)AuNPs as a catalyst.