Diverse commensal populations are now regarded as key to physiological homeostasis and protection against disease. Although bacteria are the most abundant component of microbiomes, and the most intensively studied, the microbiome also consists of viral, fungal, archael, and protozoan communities, about which comparatively little is known. Host-defense peptides (HDPs), originally described as antimicrobial, now have renewed significance as curators of the pervasive microbial loads required to maintain homeostasis and manage microbiome diversity. Harnessing HDP biology to transition away from non-selective, antibiotic-mediated treatments for clearance of microbes is a new paradigm, particularly in veterinary medicine. One family of evolutionarily conserved HDPs, β-defensins which are produced in diverse combinations by epithelial and immune cell populations, are multifunctional cationic peptides which manage the cross-talk between host and microbes and maintain a healthy yet dynamic equilibrium across mucosal systems. They are therefore key gatekeepers to the oral, respiratory, reproductive and enteric tissues, preventing pathogen-associated inflammation and disease and maintaining physiological normality. Expansions in the number of genes encoding these natural antibiotics have been described in the genomes of some species, the functional significance of which has only recently being appreciated. β-defensin expression has been documented pre-birth and disruptions in their regulation may play a role in maladaptive neonatal immune programming, thereby contributing to subsequent disease susceptibility. Here we review recent evidence supporting a critical role for β-defensins as farmers of the pervasive and complex prokaryotic ecosystems that occupy all body surfaces and cavities. We also share some new perspectives on the role of β-defensins as sensors of homeostasis and the immune vanguard particularly at sites of immunological privilege where inflammation is attenuated.