There has been growing interest in the role of viral infections and their association with adverse pregnancy outcomes. However, little is known about the impact viral infections have on the fetal membranes (FM). Toll-like receptors (TLR) are thought to play a role in infection-associated inflammation at the maternal–fetal interface. Therefore, the objective of this study was to characterize the cytokine profile and antiviral response in human FMs exposed to viral dsRNA, which activates TLR3, and viral ssRNA, which activates TLR8; and to determine the mechanisms involved. The viral dsRNA analog, Poly(I:C), induced up-regulated secretion of MIP-1α, MIP-1β, RANTES and TNF-α, and down-regulated interleukin (IL)-2 and VEGF secretion. In contrast, viral ssRNA induced a broader panel of cytokines in the FMs by up-regulating the secretion of IL-1β, IL-2, IL-6, G-CSF, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α and GRO-α. Using inhibitory peptides against TLR adapter proteins, FM secretion of MIP-1β and RANTES in response to Poly(I:C) was MyD88 dependent; MIP-1α secretion was dependent on MyD88 and TRIF; and TNF-α production was independent of MyD88 and TRIF. Viral ssRNA-induced FM secretion of IL-1β, IL-2, IL-6, G-CSF, MIP-1α, RANTES and GRO-α was dependent on MyD88 and TRIF; MIP-1β was dependent upon TRIF, but not MyD88; and TNF-α and MCP-1 secretion was dependent on neither. Poly(I:C), but not ssRNA, induced an FM antiviral response by up-regulating the expression of IFNβ, myxovirus-resistance A, 2′,5′-oligoadenylate synthetase and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G. These findings demonstrate that human FMs respond to two viral signatures by generating distinct inflammatory cytokine/chemokine profiles and antiviral responses through different mechanisms.