Biological assemblages are commonly used for assessing stream health, but there is increased interest among the freshwater research community in incorporating measures of stream function, such as metabolism, to strengthen stream-health assessments. Presently, there is limited information about the relationships between stream metabolism and biological assemblages, along with the measurement period required to relate metabolism with stream biota. Our study assessed which environmental factors explained stream metabolism and to what degree stream metabolism and minimum dissolved oxygen (DOmin) were related to invertebrate and fish metrics in streams distributed across several regions of the United States. Furthermore, we evaluated the number of metabolism monitoring days required for maximizing the ability to detect relationships between stream metabolism and biological assemblage metrics. We sampled 17 sites distributed among reference, agricultural, and urban areas for stream metabolism, nutrients, habitat, and biological assemblages (invertebrates and fishes). Overall, sites were heterotrophic with gross primary production (GPP) and ecosystem respiration (ER) related primarily to days since last high flow, canopy cover, maximum water temperature, and total phosphorus. DOmin was related to days since last high flow, canopy cover, and maximum water temperature. We were unable to determine a clear statistical relationship between invertebrate metrics (invertebrate richness; Ephemeroptera, Plecoptera, and Trichoptera richness; and scraper-taxa richness) and GPP, ER, or DOmin. In contrast, we found that 2 fish-assemblage metrics were associated with stream metabolism and DOmin. A fish multimetric index (FMMI) was negatively correlated with GPP (r = −0.5, p = 0.048) and positively correlated with DOmin (r = 0.47, p = 0.06). Percentage of omnivorous fish taxa was positively correlated with GPP (r = 0.72, p = 0.001) and ER (r = 0.55, p = 0.02) and negatively correlated with DOmin (r = −0.67, p = 0.003). The lack of detected relationships for most of the biological-assemblage metrics with stream metabolism may be partially due to 1 or more factors, including high variability, low sample size, limited range in metabolism values, assemblage metrics used, and geographic distribution of sites. Comparing stream-metabolism measurement periods (in days) to biological-assemblage metrics indicated that optimum correlations occurred at 2 d for DOmin, 3 d for GPP, and 14 d for ER. Although our study found limited relationships of stream metabolism and DOmin with biological assemblages, future studies should consider a larger sample size (≥30), 14-d or longer metabolism measurement period, and assessment of other taxa-specific or assemblage metrics.