A key challenge in managing flow alteration is determining the severity and pattern of alteration associated with the degradation of biological communities. Understanding these patterns helps managers prioritize locations for restoration and flow management actions. However, the choices made about how to use these flow-ecology relationships can have profound implications on management decisions (e.g., which biological endpoints, which thresholds, which seasonal flow components to use). We describe a process for using flow-ecology relationships to prioritize management actions that 1) Represents the most relevant components of the annual hydrograph, 2) Demonstrates an appropriate level of sensitivity in order to discriminate locations to inform decision making, 3) Aims to protect multiple biological assemblages, 4) Reduces misclassification of priority areas (i.e., error of omission). Our approach is based on the functional flows approach which uses multiple flow metrics that describe the frequency, timing, magnitude, duration, and rate of change of seasonal process-based components of the annual hydrograph. Using this approach, we performed a flow-ecology analysis of regional bioassessment data, through which we determined where flow alteration impacts biology and prioritized reaches for changes in flow management to protect aquatic resources in a highly urbanized region of southern California, where managing scarce water resources leads to difficult decisions about tradeoffs that require technical information. We identified three important functional flow metrics for each of two bioassessment indices, one based on benthic macroinvertebrates, and another based on benthic algae. Based on thresholds that describe levels of alteration as well as thresholds describing the probability of achieving a healthy biological condition, we compared nine biological threshold combinations for each index. We found instances of flow alteration that impact biological condition highly variable (0–100% of subbasins) between combinations and we present a method for finding the most appropriate combination for prioritizing locations for flow management. We apply the final thresholds to the study region and propose 16 subbasins of high priority for implementing flow management and restoration. Importantly, we show that focusing on a single biological group would result in biologically altered locations being effectively ignored.