We successfully synthesized a library of macrocyclic peptides and examined the impact of stereochemical replacements on the antimicrobial activity and cytotoxicity. Detailed analysis of the data revealed that partial or complete stereochemical substitution resulted in minimal changes in antimicrobial activity but significantly reduced cytotoxicity compared to the parent peptides. For instance, lead peptides 15c and 16c displayed similar or slightly higher MICs (1-fold increase) against all the tested bacterial strains compared to the parent p3. Despite retaining comparable antimicrobial potency, 15c and 16c exhibited substantially improved microbial selectivity over mammalian cells, as indicated by a higher therapeutic index. Timedependent killing and antibiofilm assays confirmed their membrane-disruptive properties, supported by calcein dye leakage and SEM imaging data. Furthermore, neither wild-type nor antibiotic-resistant strains of S. aureus and E. coli exhibited resistance to 15c or 16c, suggesting a multifaceted mode of action akin to naturally occurring AMPs. As expected, given the presence of multiple non-proteogenic amino acids, including D-isomers in a cyclic framework, both 15c and 16c showed good plasma stability with t1/2 of 6 h and 8 h, respectively.