Pathogens resistant to most conventional anti-infectives are a harbinger of the need to discover and develop novel anti-infective agents and strategies. Endogenous host defense peptides (HDPs) have retained evolution-tested efficacy against pathogens that have become refractory to traditional antibiotics. Evidence indicates that HDPs target membrane integrity, bioenergetics, and other essential features of microbes that may be less mutable than conventional antibiotic targets. For these reasons, HDPs have received increasing attention as templates for development of potential anti-infective therapeutics. Unfortunately, advances toward this goal have proven disappointing, in part owing to limited understanding of relevant structure-activity and selective toxicity relationships in vivo, a limited number of reports and overall understanding of HDP pharmacology, and the difficulty of cost-effective production of such peptides on a commodity scale. However, recent molecular insights and technology innovations have led to novel HDP-based and mimetic anti-infective peptide candidates designed to overcome these limitations. Although initial setbacks have presented challenges to therapeutic development, emerging themes continue to highlight the potential of HDP-based anti-infectives as a platform for next-generation therapeutics that will help address the growing threat of multidrug-resistant infections.