Grab water samples, sediment samples, and 2,2,4-trimethylpentane passive samplers (TRIMPS) were used to determine the exposure to 97 pesticides in 24 southeast Australian stream sites over 5 months. Macroinvertebrate communities and selected microorganisms (bacteria, flagellates, ciliates, amoebas, nematodes, and gastrotrichs) were sampled to detect relationships with pesticide toxicity. Sediment samples had the highest estimated toxicities in terms of toxic units (TU) for Daphnia magna (TUDM) and for Selenastrum capricornutum (TUSC). The pesticide-selective SPEARpesticides and the general SIGNAL index for macroinvertebrates exhibited negative linear relationships (r2 = 0.67 and 0.36, respectively) with pesticide contamination in terms of log maximum TUDM (log mTUDM), suggesting macroinvertebrate community change due to pesticide exposure. Pesticide contamination was the only measured variable explaining variation in ecological quality. Variation in the densities of several microbial groups was best explained by environmental variables other than log TUs. The log mTUDM values derived from sediment concentrations were most important to establish a link with effects on macroinvertebrates, whereas log mTUDM of grab water samples had only minor contribution. Current-use insecticides and fungicides can affect macroinvertebrate communities and monitoring of sediment and continuous water sampling is needed to detect these effects.