A study was undertaken for the evaluation of the pesticide pollution caused by the agricultural activities in the basin of Lake Vistonis, Greece during the years 2010-2012. Water samples were collected from Lake Vistonis, four major rivers and various small streams and agriculture drainage canals. The concentration of 302 compounds was determined after solid-phase extraction of the water samples and subsequent LC-MS/MS and GC-MS/MS analysis of the extracts. Overall, herbicides were the most frequently detected pesticides (57%), followed by insecticides (28%) and fungicides (14%). In Lake Vistonis 11 pesticides were detected. Specifically, fluometuron was detected in the 75% of the samples (maximum concentration 0.088 μg/L) whereas lambda-cyhalothrin was detected in all the samples of spring 2011 and alphamethrin in all the samples of spring 2012 (maximum concentration 0.041 and 0.168 μg/L, respectively). In the rivers and drainage canals 68 pesticides were detected. Specifically, fluometuron was detected in the 53% of the samples (maximum concentration 317.6 μg/L) followed by chlorpyrifos and prometryn (16 and 13% of the samples respectively). An environmental risk assessment was performed by employing the Risk Quotient (RQ) method. The risk assessment revealed that at least one pesticide concentration led to a RQ>1 in 20% of the samples. In Lake Vistonis, alphamethrin and lambda-cyhalothrin concentrations resulted in RQ>1, whereas in the other water bodies this was mainly the result of chlorpyrifos-methyl and alphamethrin exposure. In contrast, herbicide and fungicide concentrations contributed substantially less to environmental risks.