The misuse of antibiotics in animal protein production has driven the emergence of a range of drug-resistant pathogens, which threaten existing public health security. Consequently, there is an urgent need to develop novel antimicrobials and new infection treatment options to address the challenges posed by the dramatic spread of antibiotic resistance. Piscidins, a class of fish-specific antimicrobial peptides (AMPs), are regarded as promising therapies for biomedical applications. Progress towards potential analogs from the piscidin family has been hampered by unenforceable structural optimization strategies. Here, we leverage a strategy of bioinformatics analysis combined with molecular dynamics (MD) simulation to identify specific functional hotspots in piscidins and rationally design related analogues. As expected, this approach yields a potent and non-toxic PIS-A-1 that can be used as an antibiotic adjuvant to reverse methicillin-resistant Staphylococcus aureus (MRSA) pathogens. Remarkably, the structural optimization scheme and application strategy proposed here will contribute richer therapeutic options for the safe production of animal protein.