Commensalism coinfection of pathogens has seriously jeopardized human health. Currently, Kunitzin-RE, as an amphibian-derived bioactivity peptide, is regarded as a potential antimicrobial candidate. However, its antimicrobial properties were unsatisfactory. In this study, a set of shortened variants of Kunitzin-RE was developed by the interception of a peptide fragment and single-site mutation to investigate the effect of chain length, positive charge, hydrophobicity, amphipathicity, and secondary structure on antimicrobial properties. Among them, W8 (AARIILRWRFR) significantly broadened the antimicrobial spectrum and showed the highest antimicrobial activity (GMall = 2.48 μM) against all the fungi and bacteria tested. Additionally, W8 showed high cell selectivity and salt tolerance in vitro, whereas it showed high effectiveness against mice keratitis cause by infection by C. albicans 2.2086. Additionally, it also had obviously lipopolysaccharide-binding ability and a potent membrane-disruptive mechanism. Overall, these findings contributed to the design of short antimicrobial peptides and to combat the serious threat of commensalism coinfection of pathogens.