Urban stormwater is an important but incompletely characterized contributor to surface‐water toxicity. The present study used 5 bioassays of 2 model organisms (Daphnia magnaand fathead minnow, Pimephales promelas) to investigate stormwater toxicity and mitigation by full‐scale iron‐enhanced sand filters (IESFs). Stormwater samples were collected from major stormwater conveyances and full‐scale IESFs during 4 seasonal events (winter snowmelt and spring, early summer, and late summer rainfalls) and analyzed for a diverse range of contaminants of emerging concern including pharmaceuticals, personal care products, industrial chemicals, and pesticides. Concurrently, stormwater samples were collected for toxicity testing. Seasonality appeared more influential and consistent than site type for most bioassays. Typically, biological consequences were least in early summer and greatest in late summer and winter. In contrast with the unimproved and occasionally reduced biological outcomes in IESF‐treated and late summer samples, water chemistry indicated that numbers and total concentrations of detected organic chemicals, metals, and nutrients were reduced in late summer and in IESF‐treated stormwater samples. Some potent toxicants showed more specific seasonality (e.g., high concentrations of polycyclic aromatic hydrocarbons and industrial compounds in winter, pesticides in early summer and spring, flame retardants in late summer), which may have influenced outcomes. Potential explanations for insignificant or unexpected stormwater treatment outcomes include confounding effects of complex stormwater matrices, IESF nutrient removal, and, less likely, unmonitored toxicants.