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Surfaces submerged in an aquatic milieu are covered to some degree with biofilms - organic matrices that can contain bacteria, microalgae, and protozoans, sometimes including disease-causing forms. One unquantified risk of aquatic biological invasions is the potential for biofilms within ships' ballast water tanks to harbor pathogens, and, in turn, seed other waters. To begin to evaluate this vector, we collected biofilm samples from tanks' surfaces and deployed controlled-surface sampling units within tanks. We then measured a variety of microbial metrics within the biofilms to test the hypotheses that pathogens are present in biofilms and that biofilms have higher microbial densities compared to ballast water. Field experiments and sampling of coastwise and oceangoing ships arriving at ports in Chesapeake Bay and the North American Great Lakes showed the presence of abundant microorganisms, including pathogens, in biofilms. These results suggest that ballast-tank biofilms represent an additional risk of microbial invasion, provided they release cells into the water or they are sloughed off during normal ballasting operations.[PUBLICATION ABSTRACT]