Antimicrobial peptides (AMPs), essential components of innate immunity, are found in a range of phylogenetically diverse species and are thought to act by disrupting the membrane integrity of microbes. In this paper, we used evolutionary signatures to identify sites that are most relevant during the functional evolution of these molecules and introduced amino acid substitutions to improve activity. We first demonstrate that the anti-microbial activity of chicken avian beta-defensin-8, previously known as gallinacin-12, can be significantly increased against Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Salmonella typhimurium phoP- mutant and Streptococcus pyogenes through targeted amino acid substitutions, which confer increased peptide charge. However, by increasing the AMP charge through amino acid substitutions at sites predicted to be subject to positive selection, antimicrobial activity against Escherichia coli was further increased. In contrast, no further increase in activity was observed against the remaining pathogens. This result suggests that charge-increasing modifications confer increased broad-spectrum activity to an AMP, whilst positive selection at particular sites is involved in directing the antimicrobial response against specific pathogens. Thus, there is potential for the rational design of novel therapeutics based on specifically targeted and modified AMPs.