In the present study, three biomarkers, acetylcholinesterase (AChE) activity, vitellogenin (Vg)-like proteins, and ethoxyresorufin O-deethylase (EROD) activity, were evaluated in male Carcinus aestuarii crabs collected from four sites in the Lagoon of Venice, two in the northern lagoon area and two in the southern one. Results revealed differences among sites in relation to each specific biomarker. Hence, a suite of biomarkers can be used to discriminate sampling sites according to types of pollution, reflecting differing conditions of anthropogenic impact. Comparisons between the northern and southern lagoon areas suggest that the latter are in better condition for Vg-like protein level and AChE activity. The lower EROD activity recorded in crabs from the northern sites suggests a detrimental inhibitory effect of contaminants, rather than a lack of induction due to lower pollution levels.