Purpose: Human ocular surface epithelia express four antimicrobial peptides (APs): β -defensin (hBD) 1-3 and LL-37. Here the expression of additional APs (hBD 4-6, HE2β 1; histatin-1, -3; liver expressed antimicrobial peptide-1, -2; macrophage inflammatory protein (MIP)-3α, and thymosin (T)β -4) was sought and activity against common ocular pathogens studied. Methods: AP expression was determined in human corneal and conjunctival epithelial cells (HCEC, HCjEC) by RT-PCR and in corneal sections by immunostaining. Antimicrobial assays were performed to assess peptide (hBD 1-3, LL-37, MIP-3α, and Tβ 4) activity against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE) in the presence of NaCl or tears. Results: HCEC and HCjEC expressed MIP-3α and Tβ 4. hBD 1-3, MIP-3α, and Tβ 4 showed activity against PA. hBD-3 had potent activity against SA and SE, whereas hBD-2, MIP-3α and Tβ 4 had moderate activity and hBD-1 had none. NaCl markedly attenuated, and tears almost completely inhibited the activity of hBD 1-2 and Tβ 4, but not that of hBD-3. Conclusions: The ocular surface epithelia additionally express MIP-3α and Tβ 4 which have moderate antimicrobial activity. The current data support a role for hBD-3 as an antimicrobial peptide in vivo, but call in to question the effectiveness of some other APs. However, further study is required to conclusively elucidate the physiological role of each AP.