Background: As a new intraoperative disinfection method, chlorhexidine-wetted textile napkins have been employed in order to cover the upper and lower eyelid edges, eyelid skin, eyelashes, lid margins and palpebral conjunctiva during phacoemulsification cataract extraction. This study was conducted to compare the antimicrobial activity of textile napkins before and after their use. Methods: This study evaluated 80 textile napkins wetted with 0.02% aqueous solution of chlorhexidine. All textile napkins were divided into groups. The study group consisted of 60 used textile napkins which were collected from 29 patients (30 eyes) at the end of phacoemulsification, and the control group included 20 unused sterile textile napkins. Antimicrobial assay was performed by means of measuring the growth inhibition zones of the standard or clinical isolate strains under the textile napkins on the surface of agar media. Results: The number of textile napkins and the diameter of the growth inhibition zones (mm) in the study group and in the control group relating to gram-positive, gram-negative, and fungi were: 24/31 vs. 8/31, 32/30 vs. 8/30, and 4/30 vs. 4/30. The diameter of the growth inhibition zones of gram-positive bacteria was more than other investigated microorganisms. In the growth inhibition zones, exogenous microorganism colonies were not found. Conclusion: Antimicrobial activity of textile napkins wetted with 0.02% aqueous solution of chlorhexidine against gram-positive bacteria is more than gram-negative bacteria and fungi, and is preserved to the end of the phacoemulsification.