<p>Antimicrobial resistance poses a significant threat to global health. Host defense peptides (HDPs) are being investigated as a strategy to mitigate this threat. Porcine β-defensins (pBDs) are a class of HDPs present in the swine immune system. Our group previously identified pBD‑5 as a component of the innate immune system of pigs and found that a synthetically produced cysteine-protected analogue modulated gene expression in swine gut cells in response to <i>Brachyspira hyodysenteriae</i> infection but lacked direct antimicrobial activity. In this study, we synthesized pBD‑5 with three disulfide bonds using orthogonal protection strategies and evaluated its antimicrobial activity, structural stability at pH 7, and cytocompatibility with swine red blood cells. Circular dichroism spectroscopy revealed a secondary structure predominantly composed of β-sheets and random coils, a conformation reported for other β-defensins. Using a paired study design with three technical replicates per group, we assessed pBD-5 antimicrobial properties at eight concentrations (1.56–200 µM) against <i>Escherichia coli</i> and <i>Staphylococcus epidermidis</i>. Growth inhibition was measured by optical density (OD₆₀₀ nm) and confirmed through colony-forming unit (CFU) counts on agar plates. Dose-dependent antimicrobial activity was observed, with significant growth reductions at 100 µM for CFU assays (<i>S. epidermidis</i>: 2-log reduction, <i>P</i> = 0.0318; <i>E. coli</i>: 1-log reduction, <i>P</i> = 0.0474) and 25 µM for OD₆₀₀ nm measurements (<i>P</i> &lt; 0.001). Cytocompatibility assays revealed statistically significant hemolysis only at the concentration of 240 µM (<i>P</i> = 0.0009), highlighting the peptide’s cytocompatibility at antimicrobial concentrations. While pBD-5 demonstrated modest antimicrobial properties compared to other AMPs, its cytocompatibility suggests potential for further investigations, including in vivo experimentation.</p>