Aspergillus fumigatus is an environmental fungus that causes life-threatening infections in neutropenic patients. In the absence of intact innate immunity, inhaled A. fumigatus spores (conidia) germinate in the lung, forming hyphae that invade blood vessels and disseminate to other tissues. Although macrophages and neutrophils are postulated to provide defense against invasive fungal infection, animal models and human studies suggest that circulating monocytes also contribute to antifungal immunity. Although human monocyte subsets, defined as either CD14(+)CD16(-) or CD14(+)CD16(+), have been extensively characterized, their respective roles during fungal infection remain undefined. We isolated CD14(+)CD16(-) and CD14(+)CD16(+) monocytes from healthy allogeneic hematopoietic stem cell transplantation donors and compared their ability to phagocytose and inhibit A. fumigatus conidia. Both monocyte subsets efficiently phagocytose conidia, but only CD14(+)CD16(-) monocytes inhibit conidial germination yet secrete little TNF. In contrast CD14(+)CD16(+) do not inhibit conidial germination and secrete large amounts of TNF. Although CD14(+)CD16(-) and CD14(+)CD16(+) monocytes differ in their response to dormant conidia, responses are similar if conidia are already germinated at the time of monocyte uptake. Our study demonstrates that functional CD14(+)CD16(-) and CD14(+)CD16(+) monocytes can be isolated from allogeneic hematopoietic stem cell transplantation donors and that these subsets differ in their response to A. fumigatus conidia.