Objectives Single-chamber air cathode microbial fuel cells (MFCs) were applied as biosensors for biochemical oxygen demand (BOD) measurement of real wastewaters with considerable suspended and/or slowly biodegradable organic content. Results The measurement method consists of batch sample injection, continuous measurement of cell voltage and calculation of total charge (Q) gained during the biodegradation of organic content. Diverse samples were analyzed: acetate and peptone samples containing only soluble readily biodegradable substrates; corn starch and milk samples with suspended and colloidal organics; real domestic and brewery wastewaters. Linear regression fitted to the Q vs. BOD5 measurement points of the real wastewaters provided high (> 0.985) R2 values. Time requirement of the measurement varied from 1 to 4 days, depending on the composition of the sample. Conclusions Relative error of BOD measured in the MFCs comparing with BOD5 was less than 10%, thus the method might be a good basis for the development of on-site automatic BOD sensors for real wastewater samples. Electronic supplementary material The online version of this article (10.1007/s10529-020-03050-5) contains supplementary material, which is available to authorized users.