This study presents the synthesis and biological evaluation of novel peptide isosteres compounds aimed at developing effective antimicrobial agents. The compounds were designed, synthesized, and characterized using techniques such as nuclear magnetic resonance spectroscopy, mass spectrometry, and high-performance liquid chromatography to confirm their structural features and purity. Antimicrobial activity assays were conducted to evaluate their efficacy against a panel of pathogens, with minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) values determined. Cellular activity assays assessed cytotoxicity and selectivity towards mammalian cells. The results demonstrated varying levels of antimicrobial activity, with certain peptide isosteres exhibiting potent inhibitory effects against drug-resistant pathogens. The MIC values ranged from 6.25µg/mL to 50µg/mL, while the MBC/MFC values ranged from 12.5µg/mL to >50µg/mL. Importantly, the compounds displayed selectivity towards microbial cells, with low cytotoxicity towards mammalian cells. The findings provide insights into the structure-activity relationships of the peptide isosteres and their potential as therapeutic candidates. This study contributes to the field of peptide-based therapeutics by offering new avenues for combating antibiotic resistance and developing targeted antimicrobial therapies. The synthesized peptide isosteres hold implications for the development of alternative treatments for microbial infections, addressing the limitations of existing antimicrobial agents, and advancing the field of peptide-based research. These findings underscore the potential of peptide isosteres as promising candidates for the development of effective antimicrobial agents.