Sacrificial membranes have emerged as a promising solution to mitigate membrane fouling through their ability to be removed and regenerated while preserving membrane integrity and performance. In this study, we synthesized sacrificial trilayers on a commercial polyamide nanofiltration membrane using layer-by-layer (LbL) assembly of two-dimensional molybdenum disulfide (MoS2) nanosheets and polyelectrolytes. Quartz crystal microbalance with dissipation (QCM-D) studies showed the trilayers to be MoS2-dominant by mass and have a total areal density of 2.37 μg/cm2 after the deposition of two trilayers. The trilayer membrane showed a slight decrease in water permeability and an increase in calcium chloride rejection compared to the pristine nanofiltration membrane. The trilayer membrane demonstrated improved organic fouling resistance over the pristine nanofiltration membrane, maintaining permeability throughout six consecutive cycles. Interestingly, the trilayer membrane after gypsum scaling exhibited sacrificial layer removal and permeability recovery with simple hydraulic cleaning. This suggests the potential for membrane regeneration and sustained fouling resistance that may be achieved by utilizing disruptive interactions with the trilayer materials. In conclusion, sacrificial membranes made of MoS2 nanosheets and polyelectrolytes exhibited excellent organic and inorganic fouling resistance and promising potential for layer removal and regeneration.