Membranes and separators play an important role in microbial fuel cells (MFCs). Despite many developments of separators for electrochemical cell applications, there are barriers which limit their performance for MFCs which include ionic transfer limitations, which can increase the internal electrical resistance, and oxygen transfer, which can decrease the MFC performance. Various separator materials have been used, including ion exchange membranes (anion, cation and bipolar), microfiltration and ultrafiltration membranes, porous material using fabrics, glass fibres and polymers. Advances in the understanding of separator materials and properties have opened up opportunities to overcome these limitations, but challenges remain for their practical application. Here, a review of membrane requirements and applications in MFCs is provided and an outlook of future developments for MFC separators is presented. The working principle of MFC is followed by a discussion on potential applications of the MFC for wastewater treatment and power generation. The different membrane requirements affecting the MFC performance are discussed. The initial development of this field is much more recent than that of chemical fuel cells, with major studies occurring only in the last two decades.