AbstractExosomes show great potential in diagnostic and therapeutic applications. Inspired by the human innate immune defense, herein, we report engineered exosomes derived from monocytic cells treated with immunomodulating compounds 1α,25‐dihydroxyvitamin D3, and CYP24A1 inhibitor VID400 which are slowly released from electrospun nanofiber matrices. These engineered exosomes contain significantly more cathelicidin/LL‐37 when compared with exosomes derived from either untreated cells or Cathelicidin Human Tagged ORF Clone transfected cells. In addition, such exosomes exhibit multiple biological functions evidenced by killing bacteria, facilitating human umbilical vein endothelial cell tube formation, and enhancing skin cell proliferation and migration. Taken together, the engineered exosomes developed in this study can be used as therapeutics alone or in combination with other biomaterials for effective infection management, wound healing, and tissue regeneration.