Safety studies are essential in drug development. This study evaluates the safety of Amphotericin A21 (AmB-A21), a derivative of amphotericin B with antifungal therapeutic potential. We performed a chronic toxicity study, a targeted organ study and a dermal irritation test. To evaluate chronic toxicity, 18 male adult rats were treated orally with AmB-21 (2mg/kg) for 26weeks. The effects on body-weight and animal health were measured, and haematological, clinical chemistry and histopathological tests were conducted on various organs. In the target organ toxicity study, male adult rats received a daily oral dose of AmB-21 (2mg/kg) for 6 and 17weeks; testicle histology and testosterone levels were then evaluated. For the dermal irritation study, AmB-21 (200 and 1000mg/kg) was placed on the skin of adult male rabbits; macroscopic and microscopic studies, as well as haematological and clinical chemistry tests were then conducted. The chronic toxicity study revealed that AmB-21 caused testicle damage, and the testicle-targeted study showed structural alterations and changes in testosterone levels at 17weeks. However, these alterations were no longer observed 8weeks after discontinuation of treatment, and the testes showed very similar characteristics to those in the control group. The dermal irritation study showed skin thickening and reddening in rabbits treated with 2000mg of AmB-A21 after 14days of exposure. This same group also showed changes in liver enzymes, renal parameters and platelet levels. Based on our results, we consider AmB-21 to be a potential candidate for safe, long-term antifungal treatment given its reduced side effects.