Free-living amoebae are ubiquitous environmental protozoa found in both natural and man-made environments, including moisture-damaged buildings. Furthermore, the interaction between amoebae and bacteria has been shown to enhance the virulence and pathogenicity of some bacteria. While the inhabitants of moisture damaged buildings are known to be at risk of suffering adverse health effects, the exact causative agents and mechanisms are still obscure. To examine the possible role of amoebae in the health effects associated with moisture damages, the effects of amoebae on the cytotoxicity and proinflammatory potential of nonpathogenic microbes common in moisture-damaged buildings were investigated. First, two bacterial and three fungal strains were cultured both individually and in coculture with Acanthamoeba polyphaga. Then, mouse RAW264.7 macrophages were exposed to the cocultures as well as the individually grown bacteria, fungi, and amoebae. Finally, cell viability and production of proinflammatory mediators, i.e., nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6), were measured in macrophages after the exposure. The results revealed that cocultivation with amoebae increased the cytotoxicity of the bacterium Streptomyces californicus and the fungus Penicillium spinulosum. Moreover, the macrophages produced up to 10 times higher concentrations of NO after the exposure to these cocultures than after the exposure to individually grown microbes. Finally, the production of the cytokines was up to two orders of magnitude higher (IL-6) and up to four times higher (TNF-alpha) after exposure to the cocultures when compared to individually grown microbes. We conclude that amoebae are able to potentiate the cytotoxicity and proinflammatory properties of certain microbes associated with moisture damages.