The study aims to identify bacteria causing conjunctivitis in a central Italian area and to analyze chemosusceptibility. From 2005 to 2006, 91 conjunctival swabs were collected from acute conjunctivitis cases and screened for common bacteria and fungi. Susceptibility tests were performed on isolates. Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae amounted to 86.2%. Overall, 100% of strains were susceptible to chloramphenicol and 96.6% to quinolones. Conversely, 20.7% of isolates were tetracycline-resistant and, even if all Gram negative isolates were susceptible to gentamicin, the most frequently isolated pneumococci are constitutively resistant. The study provides support for a rational choice of empiric therapy.