The formation and entrainment of fluid mud layers in troughs of subtidal dunes were investigated in the Weser Estuary, North Sea, Germany, based on hydroacoustic measurements. Near-bed suspension layers were found to consist of a suspension of large mud flocs of variable concentration, ranging from 25 g/L below the lutocline to 70 g/L at the river bed, whereas the gelling concentration was below 70 g/L. Sites of fluid mud formation coincided with the location of the estuarine turbidity zone during slack water. On average, near-bed density gradients were initially observed in dune troughs 1.2 h before slack water, and all fluid mud layers were entrained 2.3 h after slack water. No shear instabilities occurred until 1.8 h after slack water. While the flow was oriented in the dune direction, rapid entrainment was related to the development of the turbulent flow field behind dunes and is explained to be induced by advection of strong turbulence during accelerating currents. Fluid mud layers in dune troughs were entrained at an earlier point in time after slack water, compared to adjacent layers formed on a comparatively flat bed, where dune crests did not protrude from the lutocline.