The emergence of multi-drug resistant (MDR) bacterial pathogens presents a global threat of growing importance for both Gram-negative and Gram-positive bacteria. Thus, new antibiotics overcoming current bacterial resistance mechanisms by inhibiting alternative targets are urgently needed. One promising class of compounds appear to be antimicrobial peptides produced in many organisms as part of the innate immune system to prevent infections. Here, we report our most recent efforts to improve the antibacterial activity of apidaecin 1b against human pathogens using a Quantitative Structure-Activity Relationship (QSAR) study. Thus, each of the 18 residues was substituted by all other canonical and some non-canonical amino acids. Peptides were synthesized on cellulose membranes, cleaved and their minimal inhibitory concentrations (MIC) were tested in a microdilution assay against different pathogens. Combinations of the most promising substitutions were finally tested against a panel of human pathogens. Besides improving the activity against Gram-negative bacteria, we were also able to extend the activity spectrum to Gram-positive bacteria (Staphylococcus aureus). A few lead compounds were not toxic to human cell lines and possessed reasonable serum stabilities indicating good in vivo efficacies.