We examined flow alteration-ecology relationships in benthic macroinvertebrate, fish, and crayfish assemblages in Ozark Highland streams, USA, over two years with contrasting environmental conditions, a drought year (2012) and a flood year (2013). We hypothesized that: 1) there would be temporal variation in flow alteration-ecology relationships between the two years, 2) flow alteration-ecology relationships would be stronger during the drought year vs the flood year, and 3) fish assemblages would show the strongest relationships with flow alteration. We used a quantitative richest-targeted habitat (RTH) method and a qualitative multi-habitat (QMH) method to collect macroinvertebrates at 16 USGS gaged sites during both years. We used backpack electrofishing to sample fish and crayfish at 17 sites in 2012 and 11 sites in 2013. We used redundancy analysis to relate biological response metrics, including richness, diversity, density, and community-based metrics, to flow alteration. We found temporal variation in flow alteration-ecology relationships for all taxa, and that relationships differed greatly between assemblages. We found relationships were stronger for macroinvertebrates during the drought year but not for other assemblages, and that fish assemblage relationships were not stronger than the invertebrate taxa. Magnitude of average flow, frequency of high flow, magnitude of high flow, and duration of high flow were the most important categories of flow alteration metrics across taxa. Alteration of high and average flows was more important than alteration of low flows. Of 32 important flow alteration metrics across years and assemblages, 19 were significantly altered relative to expected values. Ecological responses differed substantially between drought and flood years, and this is likely to be exacerbated with predicted climate change scenarios. Differences in flow alteration-ecology relationships among taxonomic groups and temporal variation in relationships illustrate that a complex suite of variables should be considered for effective conservation of stream communities related to flow alteration.