Flow regulation for power production and irrigation set challenges to the integrity of freshwater ecosystems. Ecological flows support a sustainable ecological status in rivers as requested by the EU Directives. This study aims to assess the impact of anthropogenic flow fluctuations on instream habitats in Nestos River (N. Greece) at a mesohabitat scale and depict a reference ecohydrological regime. Fieldwork involved mobile GPS/GIS mapping, hydromorphological measurements and fish fauna samplings by electrofishing under multiple flow conditions. In total, 81 Hydro-Morphological Units (HMUs) were surveyed and 7,532 fish individuals were recorded. Next, historical flow data (2006–2008) were introduced for upstream and downstream of Toxotes Dam. Finally, environmental and biological data were combined by implementing a MesoHABitat SIMulation model (MesoHABSIM). This software computed a habitat/flow rating curve that indicated a marginal ecohydrological status occurring at a flow range from 10 to 15 m3/s downstream Toxotes Dam. Given this, habitat suitability (optimal /suitable/unsuitable) and availability (% of Maximum Wetted Area) were mapped for all species and age classes. Moreover, Uniform Continuous Under-Threshold (UCUT) curves analysis revealed that stressful events (AQ<AQ97) occurred during summer due to water abstraction and hydropeaking. Finally, habitat Stress Days Alteration index (iSDA) indicated an increase of stressful days from 44 to 160% depending on species and age class. The findings suggest that water management policies currently applied in Nestos basin need to be revised.