· Background: Acute follicular conjunctivitis is a clinical diagnosis common to multiple etiologies, of which chlamydial infection requires specific antibiotic treatment. · Purpose: This prospective study was designed to evaluate Chlamydia trachomatis as the cause of acute follicular conjunctivitis by two sensitive tests: direct enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). · Methods: Conjunctival scrapings from patients presented with untreated acute follicular conjunctivitis were examined by ELISA and PCR, and patients were followed up for prolongation of the disease course. · Results: All 36 consecutive patients presented with acute follicular conjunctivitis were negative for Chlamydia trachomatis by ELISA and PCR. None of the patients had a prolonged course of more than 4 weeks or required treatment with systemic antibiotics as would be expected from chlamydial infection. · Conclusions: Chlamydia trachomatis was probably not responsible for the acute follicular conjunctivitis in this series, and ELISA and PCR may not be cost effective for evaluation of acute follicular conjunctivitis due to chlamydial infection. Further evaluation of the cost effectiveness of these tests is required in chronic follicular conjunctivitis.