Benthic macroinvertebrates are important components of aquatic river ecosystems. These organisms are often used for biological monitoring since they are good indicators of the aquatic freshwater environment health status which can be negatively affected by human, agricultural and industrial activities. Many studies focused on the use of observed changes in macroinvertebrate communities or populations, but studies using biochemical biomarkers in these species are almost absent. The aim of this paper was to test the employment of ecotoxicological biomarkers in Ephemeroptera larvae to assess the water quality of the Alcantara river (Sicily) from its headwater to its mouth. This river represents the main source of potable water for all the counties situated in the Alcantara valley and for the city of Messina, and is the primary irrigation source for the large surrounding agricultural lands. Acetylcholinesterase activity (AChE) and Benzo(a)pyrene monooxygenase (BPMO) activity were evaluated in Serratella ignita (Ephemerellidae) larvae from different sampling sites along the river to show the potential presence of xenobiotic substances. Environmental parameters, such as temperature, dissolved oxygen, pH, conductivity and flow velocity, were also measured at different sampling sites. The biomarker approach in invertebrates represents an early warning signal of ecotoxicological alterations, providing information on which contaminants exert toxic effects on different biota. The results showed that the highest inhibition of AChE activity in S. ignita larvae was in Randazzo and Castiglione di Sicilia villages (i.e. high and medium level of the river). Here, agricultural activities are intense, with high production of fruit, olives, citrus and the heaviest usage of fertilizers, pesticides, and neurotoxic substances like organophosphate insecticides and carbammates in all the Alcantara valley. Regarding BPMO activity, the highest value was recovered in S. ignita larvae from the river mouth, which was characterised by the highest entry of water run off of streets and loaded with xenobiotic lipophilic compounds. Both AChE and BPMO were not linked to water temperature values. The data reported in the present work represent the first attempt to monitor levels of two widely recognised enzymatic biomarkers in benthic macroinvertebrates. Results indicate the possible use of this approach in macroinvertebrate larvae to study the health status of Alcantara river. The river appears to be impacted by contamination mainly from waste discharges and, upstream, from large pasture areas. Considering the world-wide distribution of S. ignita, the biomarker approach used to assess water quality may be applicable not only to Alcantara river, but also to other lotic ecosystems.