Human-mediated vectors often inadvertently translocate species assemblages to new environments. Examining the dynamics of entrained species assemblages during transport can provide insights into the introduction risk associated with these vectors. Ship biofouling is a major transport vector of nonindigenous species in coastal ecosystems globally, yet its magnitude in the Arctic is poorly understood. To determine whether biofouling organisms on ships can survive passages in Arctic waters, we examined how biofouling assemblage structure changed before, during, and after eight round-trip military voyages from temperate to Arctic ports in Canada. Species richness first decreased (~70% loss) and then recovered (~27% loss compared to the original assemblages), as ships travelled to and from the Arctic, respectively, whereas total abundance typically declined over time (~55% total loss). Biofouling community structure differed significantly before and during Arctic transits as well as between those sampled during and after voyages. Assemblage structure varied across different parts of the hull; however, temporal changes were independent of hull location, suggesting that niche areas did not provide protection for biofouling organisms against adverse conditions in the Arctic. Biofouling algae appear to be more tolerant of transport conditions during Arctic voyages than are mobile, sessile, and sedentary invertebrates. Our results suggest that biofouling assemblages on ships generally have poor survivorship during Arctic voyages. Nonetheless, some potential for transporting nonindigenous species to the Arctic via ship biofouling remains, as at least six taxa new to the Canadian Arctic, including a nonindigenous cirripede, appeared to have survived transits from temperate to Arctic ports.Electronic supplementary materialThe online version of this article (doi:10.1007/s00227-016-3029-1) contains supplementary material, which is available to authorized users.