General anesthesia is a crucial tool in health care and clinical practice involving wildlife, including penguins. A balanced general anesthetic technique that combines multiple drugs is beneficial for achieving sufficient anesthesia while minimizing the side effects of individual agents. However, only a few studies have explored the use of multimodal anesthesia in penguins. This study examined the effects of midazolam (benzodiazepine) and butorphanol (mixed j-opioid agonist and weak l-opioid antagonist) as preanesthetic agents in gentoo penguins (Pygoscelis papua). Midazolam and butorphanol were administered intramuscularly at 0.25 mg/kg each, followed by anesthesia induction and maintenance with alfaxalone administered intravenously. Sedation level, required alfaxalone dose, recovery time, heart rate, and noninvasive blood pressure were evaluated during anesthesia. Sedation intensity increased over time after premedication. When midazolam and butorphanol were used as preanesthetics, the required alfaxalone dose for induction was 4.8 ± 0.8 mg/kg, and the mean infusion rate of alfaxalone required to maintain anesthesia was 0.12 ± 0.05 mg/kg per minute. Recovery from anesthesia took 3 (1–7) (median [interquartile range]) min for extubation and 20 ± 12 (mean ± SD) min for complete recovery. Heart rates were maintained within the normal physiological range, and noninvasive blood pressure remained stable. Compared with previous investigations on anesthesia induction and maintenance using alfaxalone alone, the intramuscular administration of 0.25 mg/kg midazolam and 0.25 mg/kg butorphanol reduced the alfaxalone dose requirement for anesthesia induction and maintenance, thereby shortening recovery times.