Small proline-rich antimicrobial peptides (AMP) have attracted considerable interest, as they target specific intracellular bacterial components and do not act by lytic mechanisms. Here, a novel peptide, termed oncocin (VDKPPYLPRPRPPRRIYNR-NH2), is reported that was optimized for the treatment of Gram-negative pathogens. Its minimal inhibitory concentrations in tryptic soy broth medium ranged from 0.125 to 8 I?g/mL for 34 different strains and clinical isolates of Enterobacteriaceae and nonfermenters, such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Substitutions of two arginine residues by ornithine increased the half-lives in full mouse serum from about 20 min to greater than 180 min and the activity. Both optimized oncocin derivatives were neither toxic to human cell lines nor hemolytic to human erythrocytes. They could freely penetrate lipid membranes and were washed out completely without any sign of lytic activity, as assessed by quartz crystal microbalance. Fluorescence labeled peptides entered the periplasmic space within 20 min at room temperature and homogeneously stained E. coli within 50 min. In conclusion, the optimized oncocin represents a very promising candidate for future in vivo work and may serve as a novel lead compound for an antibacterial drug class.