In this study, mixed culture bioelectrochemical systems were operated with various membrane separators: one prepared with 1-Butyl-3-methylimidazolium hexafluorophosphate ([bmim] [PF6]) ionic liquid and another one called Nafion, used as reference for comparative evaluation. In the course of experiments, the primary objective was to reveal the influence of membranes-type on microbial fuel cell (MFC) behavior by applying a range of characterization methods. These included cell polarization measurements, monitoring of dehydrogenase enzyme activity and cyclic voltammetry for the analysis of anode biofilm properties and related electron transfer mechanism. Additionally, MFC performances for both membranes were assessed based on Coulombic efficiency as well as substrate (acetate) concentration dependency of energy yields. As a result, it was demonstrated that the ionic liquid-containing membrane could be suitable to compete with Nafion and appears as a candidate to be further investigated for microbial electrochemical applications.