Anuran skin is known to be a rich source of antimicrobial peptides although their therapeutic potential is often limited due to their toxicity against mammalian cells. The analysis of structure-activity relationships among anuran antimicrobial peptides provided the parameters to construct the "Mutator" tool for improving their selectivity for bacterial cells, by suggesting appropriate point substitutions. Double substitution analogues [K2, K16] of the Xenopus tropicalis peptide XT-7 and [I2, K19] of the Ascaphus truei peptide ascaphin-8 were predicted by this tool to have an increased 'therapeutic index' (TI = HC(50)/MIC for erythrocytes with respect to bacteria) > 80. The mutated peptides were synthesized and respectively found to have experimental TI values > 130 for S. aureus or E. coli, a considerable improvement with respect to TI < 37 for the parent compounds. Circular dichroism studies of the mutated peptides suggested this may in part be due to variations in the α-helical structure. For P. aeruginosa, which is more resistant to XT-7, the TI increased in the mutated peptide from 5 to >270, also due to a significant improvement in minimal inhibitory concentration. We have shown that the Mutator tool is capable of suggesting limited variations in natural anuran peptides capable of increasing peptide selectivity, by decreasing toxicity against mammalian erythrocytes, in general without compromising antibacterial activity. The tool is freely available on the Mutator Web server at http://split4.pmfst.hr/mutator/.