Contraception is needed to prevent overpopulation and inbreeding in highly fecund captive bat colonies. Reports on surgical contraception in bats are limited. The objective of this study was to describe surgical castration techniques in a megachiropteran and a microchiropteran fruit bat species. Open orchiectomy by transfixing ligation of the spermatic cord was performed in 14 Ruwenzori long-haired fruit bats (Rousettus lanosus) (RL-LIG), and orchiectomy with radiosurgery alone was performed in 125 Jamaican fruit-eating bats (Artibeus jamaicensis) (AJ-RS) and one Ruwenzori bat (RL-RS). The surgical techniques were not compared in different species with the exception of the one Ruwenzori bat operated with RS. Anesthesia was induced and maintained with isoflurane in oxygen via facemask. Preoperatively, all bats received butorphanol and subcutaneous fluids. Meloxicam was administered postoperatively for the RL-LIG. For the RL-LIG, anesthesia lasted 49 ± 15 min (mean ± SD) with a total surgery time of 26 ± 12 min. In comparison, the RS was considerably shorter, lasting 10 ± 3 min for anesthesia and 5 ± 2 min for surgery. Complications were rare, with a morbidity rate of 6.7% with the RL-LIG (prolonged recovery [n = 1]) and of 4.8% with the RS (dyspnea [n = 3], hemorrhage [n = 2], and prolonged recovery [n = 1]). One of the cases of hemorrhage was in the single Ruwenzori bat castrated using the RS technique. Mortality rate was 1.6% (n = 2) with the RS. No mortality occurred with the RL-LIG. In conclusion, using radiosurgery alone appears to be a safe and rapid surgical technique in smaller species of fruit bats. For larger species, such as the Ruwenzori fruit bats, ligation or use of an advanced vessel sealing system is recommended.