Recreational and commercial navigation is omnipresent, rendering European large rivers highways for cargo vessels, passenger ships and sport boats. Any types of motorized vessels create waves and drawdown eroding shallow shore areas. Consequently, inland navigation alters the living environment of fish with specific habitat requirements on nursing, hatching and spawning along shorelines. We assess the influence of recreational (sport boats) and commercial navigation (passenger ships, cargo vessels) on fish assemblages. Seven fish population metrics (FPM) were analyzed for 396 fish samplings at 88 sites in six large rivers characterized by seven different estimates of navigation intensity to identify FPM sensitive to inland navigation. Navigation intensity was characterized by frequency, total freight transported, total carrying capacity, degree of capacity utilization and by numbers of empty running vessels, aiming to approximate whether frequency, freight or draft of cargo vessels matter most. Densities of lithophilic fish were most sensitive to frequencies of sport boats, passenger ships and cargo vessels and declined as navigation traffic increased. Densities of rheophilic fish declined likewise but were less sensitive than lithophils. Frequency, freight and carrying capacity of cargo vessels had comparable effects on FPM and are equally useful in addition to frequency of sport boats and passenger ships to assess the impacts of recreational and commercial navigation on fish assemblages. Lower species richness indicated a specific influence of vessel draft on fish diversity. Our study shows that both recreational and commercial navigation impair fish assemblages in navigable rivers. Operation-related navigation impacts act on top of river regulation and engineering works to maintain fairways in the main channel. Therefore, impacts from recreational and commercial navigation must be especially addressed in addition to mitigating impacts from river regulation and hydromorphological degradation to achieve environmental objectives such as species conservation, ecological improvements and river rehabilitation.