A series of N- and C-terminal modifications of the monomeric proline-rich antimicrobial peptide, Chex1-Arg20, was obtained via different chemical strategies using Fmoc/tBu solid-phase peptide synthesis in order to study their effects on a panel of Gram-negative bacteria. In particular, C-terminal modifications with hydrazide or alcohol functions extended their antibacterial activity from E. coli and K. pneumoniae to other Gram-negative species, A. baumannii and P. aeruginosa. Furthermore, these analogues did not show cytotoxicity towards mammalian cells. Hence, such modifications may aid in the development of more potent proline-rich antimicrobial peptides with a greater spectrum of activity against Gram-negative bacteria than the parent peptide.