The MesoHABitat SImulation Model (MesoHABSIM) is the preferred method to calculate spatio-temporal variation in the fish habitat availability in Italian rivers. With the aim of improving the applicability of the MesoHABSIM approach in the Italian territory, we carried out a systematic review of physical habitat preferences for 31 freshwater fish species and three freshwater lampreys, representing 75% of the total indigenous freshwater fish community of Italy. Information related to suitable ranges of depth, flow velocity, biotic/abiotic substrates, covers/shelters was collected and summarized for two critical life stages (adult and juvenile) and two bioperiods (rearing/growth and spawning). Overall, 250 publications were reviewed, classified as 206 peer-reviewed papers, 20 books, 7 PhD thesis, and 17 grey literature sources. Our analysis revealed substantial deficits of information about habitat requirements for more than 30% of Italian freshwater fish species. This information is particularly scarce for the most threatened endemic species, especially for their most critical bioperiod (i.e., spawning). With the aim of preserving freshwater fish biodiversity as required in the EU Biodiversity Strategy for 2030 (European Commission, 2020), accurate information on physical habitat requirements for spawning is crucial. As an example application of MesoHABSIM, the collected habitat preference information was used to define and apply mesohabitat suitability criteria for one fish species (Telestes muticellus) in a regulated river reach of Argentina Creek (Province of Imperia, Italy). This analysis demonstrates the potential for applying information from the current review to other fish species.