Although naturally occurring membrane lytic antimicrobial peptides (AMPs) and their analogs hold enormous promise for antibiotics-resistant infectious disease therapies, significant challenges such as systemic toxicities, long peptide sequences, poor understanding of structure-activity relationships, and the potential for compromising innate host defense immunity have greatly limited their clinical applicability. To improve the clinical potential of AMPs, a facile approach is adopted to design a series of short synthetic β-sheet folding peptide amphiphiles comprised of short recurring (X1Y1X2Y2)n-NH2 sequences, where X1 and X2: hydrophobic residues (Val, Ile, Phe or Trp), Y1 and Y2: cationic residues (Arg or Lys), and n: number of repeat units; with systematic variations to the cationic and hydrophobic residues to obtain optimized AMP sequences bearing minimal resemblance to naturally occurring sequences. The designed β-sheet forming peptides exhibit broad spectrum antimicrobial activities against various clinically relevant microorganisms, including Gram-positive Staphylococcus epidermidis and Staphylococcus aureus, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and yeast Candida albicans, with excellent selectivities for microbial membranes. Optimal synthetic peptides with n = 2 and n = 3 repeat units, i.e., (IRIK)2-NH2 and (IRVK)3-NH2, efficiently inhibit sessile biofilm bacteria growth leading to biomass reduction. Additionally, sequences with n = 3 repeat units effectively neutralize endotoxins while causing minimal cytotoxicities. Taken together, these findings clearly demonstrate that the rationally designed synthetic β-sheet folding peptides are highly selective, non-cytotoxic at antimicrobial levels and have tremendous potential for use as broad spectrum antimicrobial agents to overcome multidrug resistance in a wide range of localized, systemic, or external therapeutic applications.