Antibiotics can inhibit bacterial contamination and extend sperm longevity during storage; a primary goal of captive facilities conducting biobanking and artificial fertilisation (AF). This study evaluated the effects of gentamicin on the short-term storage of Booroolong frog sperm. Sperm suspensions were obtained via either testis maceration, or as spermic urine, following hormonal induction of sperm-release. The effect of 0, 1, 2, 3 or 4 mg mL−1 gentamicin on bacterial abundance (CFU mL−1) was determined and sperm motility assessed. In both testis macerate samples and spermic urine samples, gentamicin administered at intermediate-to-high doses (2, 3 & 4 mg mL−1) eliminated, or significantly reduced, bacterial abundance. Sperm samples obtained via testis maceration exhibited significantly lower sperm motility at the highest doses (3 & 4 mg mL−1). All remaining treatments (0, 1 & 2 mg mL−1) were statistically similar and maintained sperm motility >55%. Sperm samples obtained as spermic urine exhibited no difference in sperm motility or velocity when treated with gentamicin at any dose. While antibiotic treatment did not improve sperm longevity as predicted, this is the first study to demonstrate that antibiotic treatment can reduce bacterial abundance without compromising sperm motility in an anuran amphibian. Antibiotic supplementation may be an important tool for reducing pathogen transmission where sperm samples are transferred between captive institutions for biobanking and AF.