In captivity, neotropical Seba's short-tailed bats (Carollia perspicillata) (Chiroptera: Phyllostomidae) reproduce readily, and contraception can constitute an opportunity to prevent uncontrolled reproduction, which can lead to overpopulation as well as inbreeding. The authors developed a safe and efficient work flow, anesthetic, and surgical castration protocol for the reproductive control of a captive population of more than 500 bats housed at the rainforest house at the Vienna Zoo, Austria. An anesthetic protocol using 0.003 mg medetomidine per bat (approximately 0.18 mg/kg) and 0.3 mg ketamine per bat (approximately 18 mg/kg) achieved surgical anesthesia in 323/328 bats, with the remaining five requiring supplementation with isoflurane. A single bat, which had a pre-existing underlying infection, died during anesthetic induction. Standard closed orchiectomy was performed on a total of 336 bats. The practicable and safe working protocol developed required three veterinarians and 3-4 assistants for the orchiectomy of this large group of captive bats. With the described work flow, the period for castration averaged 2.38 min/bat, resulting in managing 28.2 bats/h.