The release of tributyltin (TBT) from maritime traffic represents one of the main problems of direct, diffuse, and continued contamination of the marine environment. In the present survey, the concentrations of TBT and dibultytin (DBT) in brackish waters, sediments, and the gastropods Nassarius nitidus were evaluated in order to estimate the contamination of the southern part of the Venice lagoon. TBT and DBT were determined by GC-MS/MS. Recent contamination of TBT was found in brackish waters near marinas, whereas the highest concentrations of TBT and DBT were observed in surface sediments at dockyards and harbours. High content of organotin in the gastropods sampled near the dockyards, harbours, and marinas showed a mobilisation from the sediments through the food web. The present study allowed assessment of whether, despite the ban on the use of TBT paints, waters, sediments, and biota were still being contaminated by organotin compounds in the southern Venice lagoon.