Phoneutria boliviensis (F.O.P.-Cambridge 1897) is a medically important wandering spider distributed from Central America to northern South America. This study is the first description of the natural history of this species, and presents data on several aspects of its natural history: reproductive and prey wrapping behavior, postembryonic development, and habitats in the departments of Valle del Cauca and Quindio, Colombia. Prior to copulation, the male did not engage in any courtship from a distance, but instead climbed onto the female, adopting the typical copulation position of “modern wandering spiders” (position III). Females laid up to four egg sacs; between 430–1300 hatchlings emerged after 28–34 days. After hatching, spiderlings had a third claw on all their legs and built an irregular web, where they remained until the next molt. Sexual maturity occurred after 14–17 molts, and spiders matured 300–465 days after emerging from the egg sac. The species was found in disturbed habitats associated with both dry and wet tropical forests, usually on the ground with little litter. Spiders wrapped prey in silk, moving in a stereotypically circular pattern around the prey without manipulating threads with their legs. Attachments to the substrate involved rapid movements of the anterior spinnerets, while the others remained immobile.