Currently, single TLR agonists are being utilized for vaccination and tumor immunotherapy. Here we investigated the effects of tandem combinations of TLR agonists on the production of cytokines with major focus on IFN-α, -β, -γ, TNF-α, and IL-12. Using a primary human PBMC culture system, we found that tandem combinations of TLR2–9 agonists can be inert, additive, synergistic or antagonistic. The most interesting combination was TLR2 or TLR4 agonists in combination with TLR7/8 or TLR8 agonists. TLR4–TLR7/8 combinations synergistically up-regulated IFN-γ and IL-12, enhanced IFN-α and also moderately induced TNF-α. TLR2–TLR7/8 like TLR4–TLR7/8 synergistically up-regulated IFN-γ but not IL-12. TLR9 agonist CpG2216 produced high IFN-α but failed to up regulate IFN-γ singly or in tandem. Furthermore, TLR9-induced type-1 IFN was down regulated in combination with TLR7, or TLR8 agonists. TLR3 induced significant IFN-α/-β responses when used in a complex with membrane permeability enhancer DOTAP, and additively enhanced response with agonists to TLR2, 5, 7/8, and 8. To our knowledge, this study is the first to compare cytokine responses of all the possible tandem combinations of TLR agonists in human PBMC. We identified certain combinations of TLR agonists that may or may not have advantages over single agonists, for generating an “optimal cytokine combination” preferred in combating diseases.