Hepcidin belongs to the antimicrobial peptide (AMP) family and is the key regulator of iron metabolism. It modulates iron homeostasis by binding to, and degrading the iron exporter molecule, ferroportin, thus inhibiting cellular iron efflux. Many antimicrobial peptides have a dual function; some are able to act directly as an antimicrobial agent as well as having an immunoregulatory role in the host. Toll-like receptors (TLRs) bind to components of microorganisms, activate cellular signal transduction pathways and stimulate innate immune responses. The effect of TLR3 (poly I:C) and TLR9 (CpG) co-stimulation of THP-1-derived monocytes using purified TLR ligands showed that 24 h after exposure poly I:C and CpG ligands in combination, hepcidin expression was significantly increased (10-fold) when compared to the untreated control. This combination of TLR ligands mimics simultaneous bacterial and viral infections, thus suggesting a potential key role for hepcidin in combined infections. Additionally, using a chequerboard assay, we have shown that hepcidin has an antagonistic effect in combination with the antibiotics rifampicin and tetracycline against Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, evidenced by a fractional inhibitory concentration index (FICI) > 4. This finding has important implications for future treatment regimens especially in an era of increasing antimicrobial resistance.