Peptidases are indispensable tools in biotechnology and chemical biology. However, the enzyme repertoire for the selective hydrolysis of dl-amide bonds in peptides is small. Here, we describe novel dl-peptidases that mediate complex microbial interactions. These enzymes, Lip3 and Lip7, convert lipopeptides into potent amoebicidal agents via selective dl-peptide bond cleavage. Using structural analyses and mutagenesis, we identified an unusual Ser-Lys-Lys-Tyr catalytic tetrad required for dl-specificity. Despite their high structural similarity, both enzymes show distinct substrate preferences: Lip3 acts primarily as a carboxypeptidase, removing a single C-terminal residue, while Lip7 excises a tripeptide. Although their substrate scopes are broad, they are highly specific with regard to their respective cutting sites. These features make these dl-peptidases powerful tools for elucidating the structure of complex peptide-based natural products, including tensin and WLIP. Overall, this work elucidates the molecular mechanisms of cooperative microbial defense and provides a new enzymatic toolbox for biocatalysis and natural product discovery.