In recent years novel applications of bioelectrochemical systems are exemplified by phototrophic biocathodes, biocompatible enzymatic fuel cells and biodegradable microbial fuel cells (MFCs). Herein, transparent silk fibroin membranes (SFM) with various fibroin content (2%, 4% and 8%) were synthesised and employed as separators in MFCs and compared with standard cation exchange membranes (CEM) as a control. The highest real-time power performance of thin-film SFM was reached by 2%-SFM separators: 25.7 ± 7.4 μW, which corresponds to 68% of the performance of the CEM separators (37.7 ± 3.1 μW). Similarly, 2%-SFM revealed the highest coulombic efficiency of 6.65 ± 1.90%, 74% of the CEM efficiency. Current for 2%-SFM reached 0.25 ± 0.03 mA (86% of CEM control). Decrease of power output was observed after 23 days for 8% and 4% and was a consequence of deterioration of SFMs, determined by physical, chemical and biological studies. This is the first time that economical and transparent silk fibroin polymers were successfully employed in MFCs.