Voriconazole (VRC) has activity against Aspergillus fumigatus, the most frequent cause of invasive aspergillosis in immunocompromised patients. The combination of VRC and A. fumigatus hyphae induced a more pronounced profile of expression of genes encoding inflammatory molecules in human monocytes than Aspergillus alone did. Herein, we provide further evidence of the potential mechanism underlying this immunomodulatory effect of VRC on human monocytes in response to A. fumigatus hyphae. A significant additive antifungal effect was shown when VRC was combined with monocytes against A. fumigatus hyphae. Both A. fumigatus hyphae and VRC induced pronounced profiles of mRNA and protein expression of Toll-like receptor 2 (TLR2) as well as tumor necrosis factor alpha (TNF-alpha) in THP-1 monocytic cells compared to untreated cells. The VRC-induced increase was greater than that induced by hyphae. The combination of VRC and hyphae increased mRNA and protein expression of TLR2 and TNF-alpha to even higher levels than did either VRC or hyphae alone. In contrast, TLR4 expression, both at the mRNA and protein levels, was not increased by either VRC or hyphae or their combination. In addition, significantly more NF-kappaB was translocated to the nuclei of THP-1 cells treated with VRC than untreated cells. While VRC induced more NF-kappaB than hyphae did, treatment with the combination of the two factors induced the greatest NF-kappaB expression. The pronounced profile of TLR2 signaling, TNF-alpha expression, and NF-kappaB activation in the presence of VRC suggests an immunomodulatory effect leading to a more efficient response to A. fumigatus.