Sperm-storage technologies aim to extend sperm longevity and increase the time available to achieve artificial fertilisation. The aim of the present study was to quantify the effects of antibiotic supplementation (4 mg mL–1 gentamicin) and altered gaseous storage environment (100%, 20% and 0% O2) on sperm longevity in the critically endangered booroolong frog, Litoria booroolongensis. A split-sample experimental design was adopted, whereby each sperm suspension (n = 10) was evenly divided among six experimental treatments (100% O2 with antibiotic, 20% O2 with antibiotic, 0% O2 with antibiotic, 100% O2 without antibiotic, 20% O2 without antibiotic, 0% O2 without antibiotic). Sperm suspensions were refrigerated at 5°C for the duration of the 21-day storage period. Percentage sperm motility and sperm velocity were quantified every 3 days using a computer-assisted sperm analysis system. Treatments aerated with either 100% or 20% oxygen, without the addition of the antibiotic gentamicin, consistently exhibited the highest percentage sperm motility. On Day 21 of storage, sperm suspensions in these two treatments (100% O2 without antibiotic, 20% O2 without antibiotic) maintained 61.3% and 52.0% sperm motility, respectively, whereas all remaining experimental treatments exhibited <30% sperm motility. Sperm velocity did not differ significantly among storage treatments, at any of the sampling periods, with the exception of day 21. Overall, the results from this study indicate that increased oxygen availability is beneficial to sperm longevity, but that gentamicin inhibits sperm motility in L. booroolongensis.