Purpose. An investigation into bacterial biofilm formation on and in punctal plugs. Methods. The study involved 21 patients with severe dry eye whose puncta were occluded by the use of punctal plugs. Of these, 15 had Sjogren's syndrome, 3 had non-Sjogren's syndrome, 2 had Stevens-Johnson syndrome, and 1 had graft-versus-host disease. From 17 of the 21 subjects, 18 samples of material were extracted from the holes of the punctal plugs (16 unilateral and 1 bilateral) and were subjected to enrichment culture. Nineteen punctal plugs were removed and processed for electron microscopy: 15 by scanning electron microscopy, and 4 by transmission electron microscopy. Results. Positive cultures were found in 8 of 18 (44%) samples of the material extracted from the holes of punctal plugs. In six of these eight cases (75%) the cultured bacterial species was Staphylococcus epidermidis, whereas in the other two cases (25%) it was S. aureus. In 8 of the 15 punctal plugs examined by scanning electron microscopy and in the material extracted from 1 plug that was examined by transmission electron microscopy, there was clear evidence of bacterial colonization. Conclusion. Careful observation of patients with punctal plugs is important. If material accumulates in or on a punctal plug, it may contain bacteria and may form a bacterial biofilm. In these cases, replacement of the plug, clearing of the hole, or an alternative treatment should be considered.