Alteration of hydrologic variability is considered a key pathway by which urbanization affects stream assemblages; however, understanding the mechanisms of alteration remains a challenge. One approach is to identify biological metrics that show distinct responses to flows, which can be used to assess effectiveness of flow management strategies (e.g., stormflow abstraction and infiltration). In each of six suburban, headwater streams (0.26–1.8 km²) in Cincinnati, OH (USA), we sampled continuous stream discharge, monthly baseflow water quality, and seasonal macroinvertebrate assemblages (5 times/year) from 2005–2007. Streams were dominated by isopods (Asellidae), which increased in relative proportion with increased discharge and dissolved oxygen, and lower temperatures. Richness of Ephemeroptera, Plecoptera, and Trichoptera was related to increased flows and reduced dissolved inorganic nitrogen, and therefore was not considered to be an independent measure of flow alteration. Percent burrowers were negatively related to increased discharge, responding in an opposite manner to % clingers. Nonmetric multidimensional scaling revealed distinct assemblages in April compared to July and October, which was explained by significantly higher monthly minimum discharge in the spring. In these headwater streams, seasonal variation in baseflows may be primarily responsible for structuring macroinvertebrate assemblages independently of urban-induced stormflow and water quality alteration.