Disulfide-rich macrocyclic peptides, e.g. cyclotides, represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents a major challenge. Here we report new chemo-enzymatic strategies using peptiligase variants, i.a. omniligase-1, for the efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalata B1 and variants thereof as well as of the θ-defensin RTD-1. The synthesis of the kalata B1 variant T20K was successfully demonstrated at multi-gram scale. Several ligation sites for each macrocycle render this approach highly flexible and facilitate both the larger scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering a valuable and powerful extension of the existing toolbox of enzymes for peptide head-to-tail cyclization.