The role of Toll-like receptor 9 (TLR9) in antifungal responses in the immunodeficient and allergic host is unclear. We investigated the role of TLR9 in murine models of invasive aspergillosis and fungal asthma. Neutrophil-depleted TLR9 wild-type (TLR9(+/+)) and TLR9-deficient (TLR9(-/-)) mice were challenged with resting or swollen Aspergillus fumigatus conidia and monitored for survival and lung inflammatory responses. The absence of TLR9 delayed, but did not prevent, mortality in immunodeficient mice challenged with resting or swollen conidia compared to TLR9(+/+) mice. In a fungal asthma model, TLR9(+/+) and TLR9(-/-) mice were sensitized to soluble A. fumigatus antigens and challenged with resting or swollen A. fumigatus conidia, and both groups of mice were analyzed prior to and at days 7, 14, and 28 after the conidium challenge. When challenged with resting conidia, TLR9(-/-) mice exhibited significantly lower airway hyper-responsiveness compared to the TLR9(+/+) groups. In contrast, A. fumigatus-sensitized TLR9(-/-) mice exhibited pulmonary fungal growth at days 14 and 28 after challenge with swollen conidia, a finding never observed in their allergic wild-type counterparts. Increased fungal growth in allergic TLR9(-/-) mice correlated with markedly decreased dectin-1 expression in whole lung samples and isolated dendritic cell populations. Further, whole lung levels of interleukin-17 were lower in allergic TLR9(-/-) mice compared to similar TLR9(+/+) mice. Together, these data suggest that TLR9 modulates pulmonary antifungal immune responses to swollen conidia, possibly through the regulation of dectin-1 expression.