Microbial infections have always been serious challenges to human health considering that antibiotics almost inevitably induce microbial resistance. Therefore, it is urgent to develop a new antibacterial agent that is active against drug-resistant bacteria and is less susceptible to microbial resistance. In this work, a series of host defense peptide (HDP) mimicking antibacterial poly-β-peptides were synthesized, characterized and evaluated for their biological activities. The best poly-β-peptide within this study (20 : 80 Bu : DM) displays potent and broad spectrum antibacterial activity against antibiotic-resistant super bugs and low toxicity toward mammalian cells. Moreover, these poly-β-peptides are bactericidal and kill bacteria very fast within 5 min. An antimicrobial resistance test demonstrated that bacteria develop no resistance toward the selected poly-β-peptides even over 1000 generations. Our studies demonstrate that random copolymers of heterochiral poly-β-peptides, without the need for defined secondary structures, can mimic the antimicrobial HDP. These results imply the potential application of these poly-β-peptides as new antimicrobial agents to tackle drug resistant antimicrobial infections.