Toll-like receptors (TLRs) are transmembrane proteins that are key regulators of innate and adaptive immune responses, particularly TLR4, and they have been identified as potential drug targets for the treatment of disease. Several low-molecular-weight compounds are being considered as new drug targets for various applications, including as immune modulators. Mygalin, a 417 Da synthetic bis-acylpolyamine, is an analog of spermidine that has microbicidal activity. In this study, we investigated the effect of mygalin on the innate immune response based on a virtual screening (VS) and molecular docking analysis. Bone marrow-derived macrophages and the cell lines J774A.1 and RAW 264.7 stimulated with lipopolysaccharide (LPS) were used to confirm the data obtained in silico. Virtual screening and molecular docking suggested that mygalin binds to TLR4 via the protein myeloid differentiation factor 2 (MD-2) and LPS. Macrophages stimulated by mygalin plus LPS showed suppressed gene expression of tumor necrosis factor (TNF-α), interleukine 6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as inhibition of signaling protein p65 of the nuclear factor κB (NF-κB), resulting in decreased production of nitric oxide (NO) and TNF-α. These results indicate that mygalin has anti-inflammatory potential, being an attractive option to be explored. In addition, we reinforce the importance of virtual screening analysis to assist in the discovery of new drugs.