Biological fuel cells, namely microbial desalination cells (MDCs) are a promising alternative to traditional desalination technologies, as microorganisms can convert the energy stored in wastewater directly into electricity and utilize it in situ to drive desalination, producing a high-quality reuse water. However, there are several challenges to be overcome in order to scale up from laboratory research. This study was conducted in order to better understand the performance of MDCs inoculated with marine sediments during the treatment of brackish water (5.0 g L−1 of NaCl) under three different configurations and cycles of desalination, envisaging the future treatment of saline wastewaters with conductivities lower than 10 mS cm−1. Results have shown that by increasing the desalination cycle three times, the efficiency of salt removal was improved by 3.4, 2.4 and 2.3 times for 1-MDC, 3-MDC, and 5-MDC, respectively. The same trend was observed for electrochemical data. Findings encourage further development of the MDC for sustainable brackish water and wastewater purification and future on-site utilization.