Microbial desalination cell (MDC) is a sustainable and energy-self-sufficient technology that simultaneously treats wastewater, produces electric energy, and desalinates water in a single device. The optimal desalination process depends on the potential generated in the system, which is mainly limited by cathode reaction, high external load values used, initial salinity of saline stream, or other conditions such as anolyte buffer concentration. As transport processes (i.e., migration and diffusion) in the device overlap under such conditions, it is difficult to identify the factors that affect desalination performance. Most of the existing MDC studies have used oxygen reduction (at neutral pH) as a cathode reaction limiting the performance. The removal of the cathode reaction limitation could help to clarify the operation of the MDC and identify different transport phenomena and their influence on the performance (current efficiency, freshwater production, and salt removal rate). This study presents a systematic analysis of a laboratory-scale MDC (cross-section 100 cm2, batch mode) behaviour under different initial saline concentrations from slightly brackish water (1.3 g L−1 NaCl) to seawater (40 g L−1 NaCl) without limitation in the desalination process (i.e., using a low value of external resistance and potassium ferricyanide as a liquid catholyte). For each initial salinity, the parameters of wastewater treatment capacity, energy and freshwater production are discussed and compared with the literature. The values of freshwater production between 0.5 and 10.6 L m−2 h−1 for each initial saline concentration in the saline compartment are achieved with optimal current efficiency values (80–100%). Additionally, the influence of anolyte buffer capacity on current density in the MDC system (from 0.8 to 1.2 mA cm−2) is analysed. Furthermore, the behaviour of the system during a seawater desalination process is discussed in terms of treatment capacity and Coulombic efficiency. This study could help understand the performance of these systems in possible natural saline scenarios where MDC technology can be implemented in the future.