Conventional microbial desalination cells (MDCs) can extract organic energy from wastewater for in situ utilization in saline water desalination, but are mostly unable to achieve enhanced treatment and desalination for one stream of wastewater. In this study, a multi-stage MDC (M-MDC) with two alternating anodes and cathodes was fabricated, and operated with domestic wastewater in two operational modes. In ACAC operational mode, with wastewater flowing serially from anode-1 → cathode-1 → anode-2 → cathode-2, the M-MDC realized current production of 11.4 mA and desalination efficiency of 52.4%, effluent chemical oxygen demand and total nitrogen removed 92.5% and 87.0% respectively, due to cooperative biological nitrification/denitrification and electrical migration. As a contrast in AACC mode, with wastewater flowing serially from anode-1 → anode-2 → cathode-1 → cathode-2, a higher current generation (17.2 mA) and desalination efficiency (56.4%) were achieved, due to enhanced utilization of wastewater organics in the anode chambers. The M-MDC realized simultaneous self-driven desalination and organics/nitrogen removal for the same stream of domestic wastewater, and various operation modes were proposed for enhanced wastewater treatment (ACAC mode) or energy recovery (AACC mode), indicating a promising potential for the M-MDC in simultaneous treatment and desalination of domestic wastewater.