As part of a study to develop recommendations for non-indigenous species (NIS) monitoring in Great Lakes areas at risk of invasion, we conducted intensive sampling in the Duluth-Superior Harbor and lower St. Louis River in 2005 and 2006. Of the ∼240 benthic invertebrate taxa identified, 19 were non-indigenous, including 8 first detection records for this system: New Zealand mud snail Potamopyrgus antipodarum; African/Asianorigin cladoceran Daphnia lumholtzi; Eurasian-origin amphipod Echinogammarus ischnus; Eurasian-origin bivalves Dreissena bugensis, Pisidium henslowanum and Pisidium supinum; and possibly range expanding oligochaetes Paranais frici and Pristina acuminata. Dreissenids were by far the most abundant NIS. Several other NIS were also common, but others were detected in only a few of the >200 samples taken. Non-indigenous amphipods and Dreissena were most frequently detected in sweep net and colonization plate samples of littoral vegetation, while NIS oligochaetes, gastropods, and non-dreissenid bivalves were most frequently detected in ponar and bottom sled samples of sediments. Our findings confirm that this major shipping port remains a NIS “hotspot” and emphasize that regular surveys covering a range of habitats with multiple sampling gears and thorough taxonomic effort are needed to detect and monitor non-indigenous species.