Rapid pressure fluctuations—known as hydraulic transients—occur during valve operations or load changes in turbines and pumps. The presence of biofouling, particularly caused by the golden mussel (Limnoperna fortunei), can intensify these effects and compromise the structural integrity of pressurized systems. This study experimentally evaluated the influence of such biofouling on pressure peaks during transient events in forced conduits. A hydraulic test rig was developed using PVC pipes with nominal diameters of 2½”, 3”, and 4”, tested under both clean conditions and with simulated biofouling printed in 3D, replicating mussel morphology. Results showed that, under the same initial flow rates, pressure peaks in biofouled pipes were significantly higher than in clean ones, especially in smaller diameters. To mitigate structural risks, the downstream shut-off valve closure time was modulated using a needle valve, effectively reducing peak pressures to levels closer to design limits. It is concluded that L. fortunei colonization alters transient hydraulic behavior and should be considered in the design and operation of systems vulnerable to biofouling, particularly in critical infrastructure such as water supply networks and hydroelectric power plants.