Lysine-rich peptide, designated as KABT-AMP, was designed and synthesized to supersede the irrational use of chemical antibiotics as standard therapy. KABT-AMP is a 22-amino acid helical cationic peptide (+10) and amphipathic in nature. The antimicrobial kinetics of the peptide was ascertained in the representative strains of gram-positive, gram-negative, and fungal strains, viz., Staphylococcus aureus MTCC 2940, Escherichia coli MTCC 2939, and Candida albicans MTCC 227, respectively. KABT-AMP was synthesized by solid-phase synthesis and purified using reverse-phase high-performance liquid chromatography which resulted in >95 % purity, and matrix-assisted laser desorption/ionization time of flight revealed the mass of the peptide to be 2.8 kDa. KABT-AMP showed significant broad-spectrum antimicrobial activity against the bacterial and fungal strains analyzed in the present study with survivability of 30.8, 30.6, and 31.7 % in E. coli, S. aureus, and C. albicans, respectively, at 6 h. KABT-AMP also demonstrated antibiofilm activity against the tested biofilm forming clinical isolate, Candida tropicalis. The putative membranolytic activity of the peptide was substantiated by electron microscopic analysis. Results reveal that KABT-AMP will exhibit noteworthy antimicrobial activity against multidrug-resistant bacteria and fungus at micromolar concentrations with minimal cytotoxicity and thus could be conceived for biomedical application.