Phytotoxins are naturally produced toxins with potencies similar/higher than many anthropogenic micropollutants. Nevertheless, little is known regarding their environmental fate and off-field transport to streams. To fill this research gap, a network of six basins in the Midwestern United States with substantial soybean production was selected for the study. Stream water (n = 110), soybean plant tissues (n = 8), and soil samples (n = 16) were analyzed for 12 phytotoxins (5 alkaloids and 7 phytoestrogens) and 2 widely used herbicides (atrazine and metolachlor). Overall, at least 1 phytotoxin was detected in 82% of the samples, with as many as 11 phytotoxins detected in a single sample (median = 5), with a concentration range from below detection to 37 and 68 ng/L for alkaloids and phytoestrogens, respectively. In contrast, the herbicides were ubiquitously detected at substantially higher concentrations (atrazine: 99% and metolachlor: 83%; the concentrations range from below detection to 150 and 410 ng/L, respectively). There was an apparent seasonal pattern for phytotoxins, where occurrence prior to and during harvest season (September to November) and during the snow melt season (March) was higher than that in December–January. Runoff events increased phytotoxin and herbicide concentrations compared to those in base-flow conditions. Phytotoxin plant concentrations were orders of magnitude higher compared to those measured in soil and streams. These results demonstrate the potential exposure of aquatic and terrestrial organisms to soybean-derived phytotoxins.