Escherichia coli require mediators or composite anodes for substantial outward electron transfer, >8 A/m2. To what extent non-mediated direct electron transfer from the outer cell envelope to the anode occurs with E. coli is a debated issue. To this end, the redox behaviour of non-exoelectrogenic E. coli K12 was investigated using a bi-cathodic microbial fuel cell. The electromotive force caused by E. coli biofilms mounted 0.2–0.3 V above the value with the surrounding medium. Surprisingly, biofilms that started forming at different times synchronised their EMF even when physically separated. Non-mediated electron transfer from E. coli biofilms increased above background currents passing through the cultivation medium. In some instances, currents were rather high because of a sudden discharge of the medium constituents. Mediated conditions provided similar but more pronounced effects. The combined step-by-step method used allowed a systematic analysis of exoelectronics as encountered in microbial fuel cells.