Daptomycin is a last resort antibiotic for the treatment of infections caused by many Gram-positive bacterial strains, including vancomycin-resistantEnterococcus(VRE) and methicillin- and vancomycin-resistantStaphylococcus aureus(MRSA and VRSA). However, the emergence of daptomycin-resistant strains ofS. aureusandEnterococcusin recent years has renewed interest in synthesizing daptomycin analogs to overcome resistance mechanisms. Within this context, three aromatic prenyltransferases have been shown to accept daptomycin as a substrate, and the resulting prenylated analog was shown to be more potent against Gram-positive strains than the parent compound. Consequently, utilizing prenyltransferases to derivatize daptomycin offered an attractive alternative to traditional synthetic approaches, especially given the molecule's structural complexity. Herein, we report exploiting the ability of prenyltransferase CdpNPT to synthesize alkyl-diversified daptomycin analogs in combination with a library of synthetic non-native alkyl-pyrophosphates. The results revealed that CdpNPT can transfer a variety of alkyl groups onto daptomycin's tryptophan residue using the corresponding alkyl-pyrophosphates, while subsequent scaled-up reactions suggested that the enzyme can alkylate the N1, C2, C5, and C6 positions of the indole ring. In vitro antibacterial activity assays using 16 daptomycin analogs revealed that some of the analogs displayed 2-80-fold improvements in potency against MRSA, VRE, and daptomycin-resistant strains ofS. aureusandEnterococcus faecalis. Thus, along with the new potent analogs, these findings have established that the regio-chemistry of alkyl substitution on the tryptophan residue can modulate daptomycin's potency. With additional protein engineering to improve the regio-selectivity, the described method has the potential to become a powerful tool for diversifying complex indole-containing molecules.