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The ecological integrity of rivers ultimately depends on flow regime. Flow degradation is especially prominent in Mediterranean systems and assessing environmental flows in modified rivers is difficult, especially in environments with poor hydrologic monitoring and data availability. In many Mediterranean countries, which are characterized by pronounced natural variability and low summer flows, water management actions usually focus on prescribing minimum acceptable flows estimated by hydrologic methods. In this study, a comparative assessment of environmental flow estimation methods is developed in a river with poorly monitored flows and limited understanding of past reference conditions. This assessment incorporates both a hydrologic and a fish habitat simulation effort that takes into consideration hydrologic seasonality in a Greek mountainous river. The results of this study indicate that especially in data scarce regions the utilization of biotic indicators through habitat models, may provide valuable information, beyond that achievable with hydrologic methods, for developing regional environmental flow criteria. Despite the widespread use of the method, challenges in transferability of fish habitat simulation provide undefined levels of uncertainty and may require the concurrent use of different assessment tools and site-specific study.