We report here on the structure-activity relationship study of a 14 amino acid fragment of the cathelicidin-related antimicrobial peptide (CRAMP), CRAMP20–33 (KKIGQKIKNFFQKL). It showed activity against Escherichia coli and filamentous fungi with IC50 values below 30 μM and 10 μM, respectively. CRAMP20–33 variants with glycine at position 23 substituted by phenylalanine, leucine or tryptophan showed 2- to 4-fold improved activity against E. coli but not against filamentous fungi. Furthermore, the most active single-substituted peptide, CRAMP20–33 G23 W (IC50 = 2.3 μM against E. coli), showed broad-spectrum activity against Candida albicans, Staphylococcus epidermidis and Salmonella Typhimurium. Introduction of additional arginine substitutions in CRAMP20–33 G23 W, more specifically in CRAMP20–33 G23 W N28R or CRAMP20–33 G23 W Q31R, resulted in 3-fold increased activity against S. epidermidis (IC50 = 4 μM and 4.8 μM, respectively) as compared to CRAMP20–33 G23 W (IC50 = 15.1 μM) but not against the other pathogens tested. In general, double-substituted variants were non-toxic for human HepG2 cells, pointing to their therapeutic potential.