Abstract: Microbial desalination cells (MDCs) have been widely used to treat high-salinity wastewater. It is often required for the desalination efficiency with less membrane fouling and system scalability. In this study, a five-chamber MDC (FMDC) configuration was designed and constructed to treat the dairy wastewater. Two anode chambers and two cathode chambers were arranged symmetrically to form a central “cross-shaped” desalination channel. The effective volume of the desalination chamber was expanded to 500 mL. Single-batch treatment was enhanced to optimize the ion migration pathways. A two-stage experimental strategy was implemented to evaluate the performance. 1) A parallel comparative experiment was conducted on the FMDC and a conventional three-chamber MDC (TMDC) using synthetic wastewater. The performance differences between configurations were clarified after comparison. 2) Coagulant-pretreated dairy wastewater was taken as the treatment group (Trt), and a NaCl solution with a similar total dissolved solids (TDS) concentration was used as the control (Crt). A systematic investigation was made to explore the bioelectricity generation, desalination efficiency, organic degradation, multi-ion migration behavior, and membrane fouling in the FMDC system. The results demonstrated that the mass desalination rate of the FMDC reached 18.25 mg/(L·h), which was 2.61 times that of the TMDC (7.00 mg/(L·h)), while the output voltage showed no significant difference. Furthermore, the FMDC maintained a high desalination rate of 98.1% and stable operating voltage, even when the treatment volume was expanded by five times. The promising potential was provided for the efficient desalination and scale-up. The FMDC also exhibited a stable treatment for dairy wastewater. In electricity generation, the maximum output voltage and maximum power density were 593.60 mV and 874 mW/m², respectively. The current output was sustained lower level than the control group with the synthetic brine. In pollutant removal, a chemical oxygen demand (COD) removal rate exceeded 78% with the desalination rate above 86% and the maximum instantaneous desalination rate of 39.58 mg/(L·h). The performance was significantly constrained by the complex real wastewater. Electrochemical impedance analysis revealed that the internal resistance increased by approximately 60.57% with the real wastewater, compared with the control. The resistance further rose to 298 Ω after long-term operation. This was primarily attributed to membrane scaling and organic-biological composite fouling induced by multivalent ions (e.g., Ca²⁺, and Mg²⁺) in the wastewater. Scanning electron microscopy (SEM) confirmed that the dense fouling layer was formed to directly reduce 25.60% the average desalination rate (15.32 vs. 19.31 mg/(L·h)) with the prolonged desalination cycle. Furthermore, the ion migration analysis indicated that the removal rates were varied in the different ions, due to the migration competition, catholyte back-diffusion, and anodic microbial metabolism. For instance, the sulfate (SO₄^2-) removal rate was 96.00%, whereas the potassium (K⁺) removal rate was only 57.59%. Notably, the membrane fouling was partially reversible, as the chemical cleaning restored the transmembrane recovery rates of Ca²⁺ and Mg²⁺ to 89.35% and 79.49%, respectively. The FMDC is feasible to treat the high-salinity complex dairy wastewater using the dual configuration, in terms of the desalination rate and treatment capacity. More importantly, the membrane fouling can be identified as the limiting factor in engineering applications. Consequently, this finding can provide a significant configuration and optimization for the MDC technology on the saline wastewater. A crucial theoretical and practical guidance can also be offered for the subsequent antifouling materials under the cleaning strategies.