Intraocular levels of ofloxacin are documented after topical and systemic administration, but systemic administration of ofloxacin in ocular compression has not yet been studied. This study was undertaken to determine the intraocular penetration of systemic ofloxacin into aqueous and vitreous humor after the application of ocular compression in rabbit eyes. Ocular compression with the Honan balloon was applied for 30 min to the right eyes of 11 albino New Zealand white rabbits. After the application of ocular compression, 2 mg/mL of ofloxacin was administered intravenously. Samples from aqueous and vitreous humor were collected 30 min after infusion. Ofloxacin concentrations were determined through high-performance liquid chromatography. The mean aqueous level of ofloxacin was significantly higher in the compression group (2.40±1.00 gmg/mL) than in the no-compression group (1.61 ±1.06 μg/mL) (P < .05). The mean vitreous concentrations of ofloxacin were 0.70+-0.33 μg/mL and 0.50±0.18 μg/mL in the compression and no-compression groups, respectively. A significant difference was observed between vitreous levels of ofloxacin in the compression and nocompression groups (P < .05). Ocular compression enhanced the penetration of ofloxacin in both aqueous and vitreous humor. The drug level in the aqueous humor was sufficient for the minimum inhibitory concentration for 90% of isolates (MIC90) to inhibit most microorganisms. Although the mean vitreous ofloxacin concentration was increased by previous ocular compression, it was not sufficiently above the MIC90 for most ocular pathogens that caused endophthalmitis.