The emission of engineered nanoparticles (ENPs) into the environment in increasing quantity and variety raises a general concern regarding potential effects on human health. Compared with soluble substances, ENPs exhibit additional dimensions of complexity, that is, they exist not only in various sizes, shapes and chemical compositions but also in different degrees of agglomeration. The effect of the latter is the topic of this review in which we explore and discuss the role of agglomeration on toxicity, including the fate of nanomaterials after their release and the biological effects they may induce. In-depth investigations of the effect of ENP agglomeration on human health are still rare, but it may be stated that outside the body ENP agglomeration greatly reduces human exposure. After uptake, agglomeration of ENPs reduces translocation across primary barriers such as lungs, skin or the gastrointestinal tract, preventing exposure of “secondary” organs. In analogy, also cellular ENP uptake and intracellular distribution are affected by agglomeration. However, agglomeration may represent a risk factor if it occurs after translocation across the primary barriers, and ENPs are able to accumulate within the tissue and thus reduce clearance efficiency.