This paper provides an updated account of the occurrence and abundance of non-indigenous species (NIS) in an area of high risk of introduction: the Lagoon of Venice (Italy). This site is a known hotspot of NIS introductions within the Mediterranean Sea, hosting all the most important vectors of introduction of marine NIS—shipping, recreational boating, shellfish culture and live seafood trade. The recent literature demonstrates that the number of NIS in Venice is continuously changing, because new species are being introduced or identified, and new evidence shows either an exotic origin of species previously believed to be native, or a native origin of formerly believed “aliens”, or demonstrates the cryptogenic nature of others. The number of NIS introduced in the Venetian lagoon currently totals 71, out of which 55 are established. This number exceeds those displayed by some nations like Finland, Portugal or Libya. Macroalgae are the taxonomic group with the highest number of introduced species (41 % of NIS): the most likely vector for their introduction is shellfish culture. The source region of NIS introduced to Venice is mainly represented by other Mediterranean or European sites (76 %). The Lagoon of Venice represents a sink but also a source of NIS in the Mediterranean Sea, as it is the site of first record of several NIS, which have since further spread elsewhere.