Background: Recently, retrobulbar injection of liposomal amphotericin B has been explored as an alternative treatment of rhino-orbital-cerebral mucormycosis. Objectives: This study aims to measure amphotericin B concentration in the periocular fat tissue following intravenous, retrobulbar, and combined intravenous and retrobulbar injections. Methods: In this study, 45 rats were divided into 15 groups, receiving either intravenous, retrobulbar, or combined intravenous and retrobulbar injections. Three groups received the same dose of liposomal amphotericin B. Rats were sacrificed at 4-, 6-, and 24-hours post-injection and the periocular fat tissue was analyzed for amphotericin B concentration using HPLC. Results: Results showed that amphotericin B concentrations after intravenous injection of 10 mg/kg were 0.0001, 0.1154, and 0.0693 μg/mL at 4, 6, and 24 hours, respectively; for 15 mg/kg, the concentrations were 0.0339, 0.3534, and 0.4209 μg/mL. Retrobulbar injection resulted in concentrations of 8.8965, 9.8124, and 9.4156 μg/mL. Combined injections (10 mg/kg IV + 0.25 mg/kg retrobulbar) yielded concentrations of 8.8401, 7.8869, and 8.6409 μg/mL, while the combined 15 mg/kg IV + 0.25 mg/kg retrobulbar yielded 8.1940, 8.5277, and 9.0889 μg/mL. Conclusions: The findings indicate that retrobulbar injection of liposomal amphotericin B achieves suitable drug concentrations in periocular tissue, suggesting that for rhino-orbital-cerebral mucormycosis, retrobulbar injection alone may be sufficient, potentially eliminating the need for intravenous administration.