The acidic lipopeptides, including the clinically approved antibiotic daptomycin, constitute a class of structurally related branched cyclic peptidolactones and peptidolactams synthesized by nonribosomal peptide synthetases (NRPSs). In this study, the excised peptide cyclases from A54145 and daptomycin NRPSs were shown to be able to catalyze the macrocyclization of peptide thioester substrates, which were chemically produced by solid phase peptide synthesis. Applying this chemoenzymatic strategy, we generated derivatives of A54145 and daptomycin as well as hybrid molecules of both compounds. Bioactivity determination of the derived cyclic molecules revealed new insights into the structure-activity relationship of the acidic lipopeptide family. The general importance of several amino acid positions, including two conserved aspartic acid residues, was confirmed to be substantial for antibiotic potency. As a robust macrocyclization catalyst, the peptide cyclase excised from A54145 synthetase is the first cyclase of a branched cyclic lipopeptide, which catalyzes both macrolactonization and macrolactamization. The results presented herein illustrate the advantages of combining organic synthesis with natural product biosynthetic enzymes to explore the interplay between structural features and biological activity.