Proline-rich antimicrobial peptides (PrAMPs) are short peptides naturally synthesized by arthropods and mammals. According to their ability to inhibit translation, PrAMPs are classified into two subgroups. While type I PrAMPs arrest ribosomes at start codon of open reading frames, type II PrAMPs apidaecin (Api) and drosocin (Dro) stall ribosomes at stop codons. Notably, Api and Dro share sequence similarities specially at their C-termini, which include amino acids critical for target interaction. In this work, we bioinformatically identified Dro-like peptides encoded in the genomes of diverse fruit fly species. By testing the antimicrobial activity of the synthetic version of ten of these peptides, we found that only two of them (besides the already characterized Dro from D. melanogaster) were able to kill the tested Gram-negative bacteria. Further, the identified active peptides had the ability to cause stop codon readthrough in E. coli cells and cause ribosome arrest at stop codons during in vitro translation. Intriguingly, the peptides unable to penetrate and kill the tested strains arrested ribosomes at start codons, resembling the behavior of type I PrAMPs. The outcomes of our study underscore the complexity of predicting MOA solely based on similarity of peptide sequences.