Water pollution is a major environmental and human health issue and the Sarno river is regarded as the most polluted River in the whole of Europe. This study aimed at evaluating antioxidant response to freshwater pollution in the Sarno River (Campania, South Italy), using bags containing the biosensor water plant, Lemna minor L. Homogeneous samples of L. minor, collected from the Botanical Gardens of the University of Naples, were accurately washed and disposed in nylon bags. The bags were exposed for one week, floating on the water of the Sarno River at three sites, to determine antioxidant response to pollutants. Five heavy metal concentrations, representative of pollution of the River, were measured in the three exposure sites. Ultrastructural alterations of the exposed samples were examined to confirm the environmental stress, which underwent the exposes samples. Three different methods were used to measure the antioxidant activity: Trolox equivalent antioxidant capacity, chemiluminescence, and GST activity assays. Moreover, some specimens were in vitro exposed to the same heavy metal (HM) concentrations measured in the three exposure sites of the River and the results were compared with those obtained from the bag experiment. L. minor reacted to pollution increasing the antioxidant activity, which gets higher from the river springs towards the flatland (see bag experiments) and is related to the heavy metal concentrations (see in vitro experiments). Furthermore, TEM observations showed that cell ultrastructure alterations increased along the river in the same way as the antioxidant activity. The results confirmed the potential use of this species as biosensor and this methodological approach both in biomonitoring and phytoremediation studies.