The ichthyocidal activity of <i>Pfiesteria piscicida</i> dinospores was examined in an aquarium bioassay format by exposing fish to either <i>Pfiesteria</i>-containing environmental sediments or clonal <i>P. piscicida</i>. The presence of <i>Pfiesteria</i> spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within "cages" of variable mesh sizes were killed in those where the pore size exceeded that of <i>Pfiesteria</i> dinospores. In vitro exposure of fish larvae to clonal <i>P. piscicida</i> indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of <i>Pfiesteria</i> spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of <i>Pfiesteria</i> spp. and (ii) that the ichthyocidal activity of <i>Pfiesteria</i> spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.Instituto de Limnología "Dr. Raul A. Ringuelet"Facultad de Ciencias Naturales y Museo