θ-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC50 = 390 ± 20 nM, Ki = 365 ± 20 nM) and a weak inhibitor of other mammalian metalloproteases such as TNFα converting enzyme (TACE) (Ki = 4.45 ± 0.48 μM). Using full sequence amino acid scanning in combination with a highly efficient "one-pot" cyclization-folding approach, we obtained an RTD-1-based peptide that was around 10 times more active than wild-type RTD-1 in inhibiting LF protease (IC50 = 43 ± 3 nM, Ki = 18 ± 1 nM). The most active peptide was completely symmetrical, rich in Arg and Trp residues, and able to adopt a native RTD-1-like structure. These results show the power of optimized chemical peptide synthesis approaches for the efficient production of libraries of disulfide-rich backbone-cyclized peptides to quickly perform structure-activity relationship studies for optimizing protease inhibitors.