Agricultural use of organophosphate pesticides are responsible for surface water toxicity in California and has led to a number of impaired water body listings under section 303(d) of the Clean Water Act. Integrated passive-treatment systems can reduce pesticide loading in row crop runoff, but they are only partially effective for the more soluble organophosphates. The Landguard™ enzyme has been effectively proven as an on-farm management practice for the removal of chlorpyrifos and diazinon in furrow runoff, but it has not been used in larger-scale treatment because of concerns regarding the potential impact on in-stream macroinvertebrates after chronic use. A first-order agricultural creek was treated with the Landguard enzyme for 30 days approximately 450 m upstream of its intersection with the Salinas River. Toxicity and pesticide chemistry were measured in the creek during treatment as well as in the river both upstream and downstream of the creek input before and after treatment. Benthic macroinvertebrates were also surveyed in the river before and after enzyme treatment. Low concentrations of organophosphate pesticides were detected in the creek, but Landguard removed detected concentrations of chlorpyrifos. Toxicity detected in the creek was likely caused by pyrethroid pesticides, and no toxicity was detected in river samples. There were no differences in habitat or macroinvertebrate assemblages between upstream and downstream samples or between pre- and post-treatment samples. These results indicate that chronic treatment of the creek with Landguard enzyme had no impact on macroinvertebrate community structure in the river.