Nineteen adult, free-ranging Macquarie River turtles (Emydura macquarii) were used to examine the effects of IV and IM dosing of alfaxalone. Turtles were randomly assigned to one of two treatment groups, and alfaxalone at a dose of 9 mg/kg was injected into either the right jugular vein (n = 10) or left thigh musculature (n = 9) of individual animals. Heart rate, respiratory rate, cloacal temperature, time to regaining spontaneous movement, and response to noxious stimuli were recorded. Intravenous alfaxalone at a dose of 9 mg/kg was an effective sedative in all turtles within this treatment group and allowed for rapid intubation with no obvious complications. Mean sedation time for turtles given alfaxalone IV was 14.3 min (SD: 5.4, min–max: 8–25). All turtles administered IV alfaxalone underwent a prolonged period of reduced respiratory rate, but all recovered uneventfully. Turtles within the IV group also maintained withdrawal reflexes, palpebral reflexes, and jaw tone for the duration of the experiment and, as such, alfaxalone must be used in conjunction with other methods of analgesia under circumstances where pain is expected to occur. Conversely, IM alfaxalone at a dose of 9 mg/kg did not result in any sedative effects and was therefore considered ineffective.