The purpose of this study was to determine normal ocular surface bacterial flora, tear production, and intraocular pressure in a captive flock of Humboldt penguins, Spheniscus humboldti. Twenty-eight healthy penguins were studied and equally divided between fresh- and saltwater habitats. The population consisted of 15 female and 13 male penguins, ranging from 3–20 years of age. Following complete ophthalmic exam, 4 penguins with cataracts were removed from the study. Eight penguins from each habitat were randomly selected for ocular surface aerobic bacterial culture. Corynebacterium spp. and Staphylococcus spp. were the most common isolates. Twenty-five organisms consisting of 17 species, and 15 organisms consisting of 9 species, were identified in fresh- and saltwater groups, respectively. Tear production and intraocular pressures were evaluated on 24 penguins with normal ocular exams. The range and mean (±standard deviation) tear production, measured with the Schirmer tear test, was 1–12 mm/min and 6.45 mm/min ± 2.9, respectively. The mean tear production for penguins housed in the freshwater habitat was greater (8.5 mm/min) than those in saltwater (4.8 mm/min). The range and mean (±standard deviation) intraocular pressure, measured by applanation tonometry using a Tono-Pen XL® tonometer, was 10–27 mmHg and 20.36 mmHg ± 4.1, respectively. This data should be utilized as a reliable resource for those involved in avian and zoo medicine.