Agriculture within the Great Barrier Reef catchment area has contributed to pesticide contamination of adjacent freshwater ecosystems that flow into the Great Barrier Reef World Heritage Area. A novel multispecies toxicity test was used to assess the toxicity of diuron and hexazinone, 2 herbicides commonly detected within the Great Barrier Reef catchment area, to a community of 3 tropical freshwater microalgae: Monoraphidium arcuatum, Nannochloropsis-like sp., and Pediastrum duplex. Diuron was the most toxic herbicide, with 10% inhibition concentration (IC10) values of 4.3, 7.1, and 29 µg/L for P. duplex, M. arcuatum, and Nannochloropsis-like sp., respectively, followed by hexazinone, with IC10 values of 15, 18, and 450 µg/L, respectively Toxicity testing on 2 commercial formulations (Barrage, 13.2% hexazinone and 48.6% diuron; Diurex, 90% diuron) showed that additives in the commercial formulations did not significantly increase the toxicity of diuron. Direct toxicity assessments were carried out on water samples from the herbicide-contaminated Sandy Creek, which discharges to the Great Barrier Reef lagoon, and a clean reference site, Tully Gorge in the Tully River. Toxicity was observed in several Sandy Creek samples. Artificial herbicide mixtures were assessed in synthetic soft water and natural freshwaters, with toxic responses being observed at environmentally relevant concentrations. The present study successfully applied a novel multispecies tropical microalgal toxicity test, indicating that it is an effective tool for the assessment of herbicide toxicity in both natural and synthetic freshwaters. Environ Toxicol Chem 2021;40:473–486. © 2020 SETAC.