Calcium (Ca2+) plays an important role in guaranteeing the formation and structure of microbial biofilm. However, little is known regarding its influence on the structure and electron transfer properties of electroactive biofilms (EABs). In this study, advanced investigative techniques such as microscopy and electrochemistry are employed to characterize biofilm structure and electron transfer properties following growth at different Ca2+ concentrations in a bioelectrochemical system (BES). As revealed by electrochemical measurements, a lower current is obtained from the EABs formed in the presence of higher Ca2+ concentrations. The decline of the current is due to the dominance of non-active cells and non-exoelectrogens in the biofilms formed at high Ca2+ concentrations. The results of this study provide a better understanding of the effect of Ca2+ on EABs structure and thus electricity generation in BESs. (C) 2015 Elsevier B.V. All rights reserved.