We have developed small synthetic cyclic amphiphilic AMPs and have reported a series of cationic macrocyclic peptides composed of various natural and unnatural amino acid residues, demonstrating promising antibacterial activity as well as high enzymatic stability. Our previous investigations explored the number and respective positions of cationic and hydrophobic residues, providing valuable insights into structure-activity relationships (SAR) in cyclic peptides and offering guidance for designing novel macrocyclic scaffolds with favorable structural properties. In this study, to further improve key therapeutic properties in addition to efficacy, such as selectivity and stability, we are reporting a new series of cyclic peptides designed by systematically replacing the cationic and/or hydrophobic residues with their respective Disomers. All synthesized cyclic peptides were screened against a wide range of Gram-positive and Gram-negative bacteria and fungi, including drug-resistant strains. In vitro cytotoxicity of the peptides was evaluated by measuring the cell viability against different types of normal mammalian cells and lytic activity against human red blood cells (hRBCs). The bactericidal and fungicidal kinetics and biofilm disruption potential of the lead cyclic peptides were measured and compared with the standard antibiotics. The proteolytic stability and potential of lead cyclic peptides against resistant and non-resistant bacteria were determined. We also examined the membrane perturbation action of the lead peptides by conducting calcein dye leakage assay, with further confirmation by scanning electron microscopy (SEM). A detailed analysis of structural, dynamic, and hydrophobic properties of lead cyclic peptides was conducted using Nuclear Magnetic Resonance (NMR) spectroscopy and molecular modeling in aqueous solution and in water-lipid environments that mimic bacterial and mammalian membranes. Altogether, the activity results of the lead cyclic peptide, along with the interaction studies in the presence of model phospholipid bilayer membranes, provided insight into the peptides' membrane disruptive mechanism.