Introduction: The round window membrane (RWM) acts as a pressure-relieving membrane for incompressible cochlear fluid. The reinforcement of the RWM has been used as a surgical intervention for the treatment of superior semicircular canal dehiscence and hyperacusis. The aim of this study was to investigate how RWM reinforcement affects sound pressure variations in the cochlea. Methods: The intracochlear sound pressure (ICSP) was simultaneously measured in the scala tympani (ST) and scala vestibuli (SV) of cadaveric human temporal bones (HTBs) in response to acoustic stimulation for three RWM reinforcement materials (soft tissue, cartilage, and medical-grade silicone). Results: The ICSP in the ST was significantly increased after RWM reinforcement for frequencies below 2 kHz. Between 400 and 600 Hz, all three materials demonstrated the highest median pressure increase. The higher the RWM stiffness, the larger the pressure increase: silicone (7 dB) < soft tissue (10 dB) < cartilage (13 dB). The ICSP in the SV was less affected by reinforcement. The highest median pressure increase was 3 dB. The experimental findings can be explained with numerical models of cochlear mechanics. Discussion and conclusions: RWM reinforcement increases the sound pressure in ST at lower frequencies but only has a minor influence on the SV pressure.