The effects on the microtidal lagoon of Lesina of runoff and the discharge of water and material from agricultural activities were investigated combining chemical analyses of pollutants [11 metals and 16 priority polycyclic aromatic compounds (PAHs)], determination of organic matter and grain size, and performance of innovative ecotoxicological tests. For metals, enrichment factors >3 for arsenic, nickel, and copper (Cu) were observed in the eastern zone of the lagoon, which is affected by nearby urban activities with discharge of water and domestic waste and by agricultural input with waters rich in fertilizers. Cu was correlated with no other metal, and its high concentrations (≤77 µg g(-1)) may result from the use of Cu-based fungicides in vineyards. Total PAHs (2,230 ± 3,150 ng g(-1)) displayed a wide range of concentrations with hot spots near freshwater inputs from the part of the catchment area exploited for wheat crops. Pyrolitic contamination also emerged, with higher-mass PAH congeners, such as asphalt, bitumen or coal, usually present in higher fractions as the dominant components. Ecotoxicological evaluations recorded moderate to high toxicity levels; the innovative MOT test bioassay showed good discriminatory ability because it identified a lagoon area whose inputs mainly depend on agricultural activities and which is impacted by metals rather than PAHs. Floods during periods of heavy rain and the discharge of water and material from agricultural activities may impact vulnerable systems, such as the lagoon of Lesina, where the presence of hot spots with remarkably high pollution values was observed.