The AltEn bioenergy plant (Mead, Nebraska, USA) previously (2015 to 2021) generated ethanol from almost 100% unused/expired treated corn seeds. This use of treated corn seeds as a feedstock for ethanol production resulted in accumulation of large amounts of contaminated wastewater and solid residue at the plant, a portion of which was applied to surrounding farmland. To better understand the potential long-term environmental effects from the processing of treated seeds, five nearby stream sites were sampled from March to July 2022 after the closure of the AltEn plant. Water and bed sediment were collected in March (prior to crop planting) through July and algae, fish, and insect samples were collected in July. Overall, 60 pesticide compounds (parents and transformation products) were detected across all matrices. In surface water samples, 52 pesticides were detected with thiamethoxam degradate (NOA-407475) having the highest concentration of 4556.2 nanograms per liter (ng/L). Algae samples had 19 pesticides detected with atrazine having the highest concentration of 191.37 nanograms per gram dry weight (ng/g d.w.). Bed sediment (bottom material) had a total of 15 pesticides detected with the highest concentration of 2.30 ng/g d.w. of acetochlor. Insect samples had 14 pesticides detected with the highest concentration of 116.10 ng/g d.w. of clothianidin. The last matrix, fish, had a total of 5 pesticides detected with the highest concentration being for atrazine of 2.21 ng/g d.w.