Mammalian β-defensins are small cationic peptides of approximately 2-6 kDa that have been implicated in mediating innate immune defenses against microbial infection. This present study investigated the activity of mouse β-defensin 3 (MBD3) against bacterial and yeast drug-resistant strains in vitro, and whether this molecule acts in synergy with antibiotics. Minimum inhibitory concentrations (MICs) and minimum bactericidal/fungicidal concentrations (MBC/MFC) of recombinant MBD3 (rMBD3) were determined by microdilution assays against different strains of Staphylococcus aureus and Candida albicans. rMBD3 inhibited the growth of S. aureus (MIC, 25 μg/ml) and C. albicans (MIC, 25 μg/ml), and showed fungicidal activity against this yeast (MFC, 100 μg/ml). The influences of rMBD3 on S. aureus and C. albicans cells were examined using electron microscopy. Cells treated with rMBD3 showed morphological and structural changes, including delamination and perforation of the peripheral cell walls, porosity, and inanition of the cytoplasmic contents. Synergistic activities of rMBD3 with different antibiotics were assessed using checkerboard tests. Interestingly, the anti-methicillin-resistant S. aureus activity of rMBD3 in combination with ampicillin was synergistic; however, this was not the case against S. aureus (ATCC 25923). Combinations of rMBD3 with itraconazole, amphotericin or 5-fluorocytosine were synergistic against the two tested C. albicans strains. These results support the interest devoted to defensins as a novel class of antimicrobial agents, and highlight their abilities to potentiate the activities of conventional antibiotics.