PURPOSE. To investigate the biofilm-forming capacity of Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa from ocular infections. METHODS. S. epidermidis strains, S. aureus strains, and P. aeruginosa strains were isolated from patients with ocular infections between 2009 and 2011. The biofilm-forming capacity of these bacteria was examined using Congo red agar (CRA) and microtiter plate assays. The biofilm-forming related genes, icaA, of S. epidermidis and S. aureus, and pslA, of P. aeruginosa, were detected using PCR. Additionally, the morphology of biofilms was observed using a scanning electron microscopy (SEM). RESULTS. Of the isolated S. epidermidis strains, 34.38% were CRA positive, 28.13% were adherence positive using the microtiter plate assay, and 40.63% carried the icaA gene. Of the isolated S. aureus strains, 55.56% were CRA positive, 51.90% were adherence positive, and 11.11% carried the icaA gene. None of the P. aeruginosa strains were phenotypic positive using CRA and microtiter plate assays, whereas 31.03% contained the pslA gene. There were significant differences between the three species when the biofilm-forming capacity of the strains was compared using the CRA method (P < 0.01) and the microtiter plate assay method (P < 0.01). CONCLUSIONS. Ophthalmic isolates of S. epidermidis and S. aureus could produce biofilms in vitro, whereas clinical strains of P. aeruginosa could not. The bacterial strains possess the genetic ability to produce biofilms, but that does not necessarily mean that biofilms will be produced. The knowledge of the process of bacterial adhesion suggests that a timely and appropriate intervention strategy be implemented in the early stages of biofilm-mediated infections. (Invest Ophthalmol Vis Sci. 2012;53:5624-5631) DOI:10.1167/iovs.11-9114