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O-carboxymethylchitosan films containing tetracycline were prepared by the casting method. The films were hardened by reaction with glutaraldehyde-induced crosslinking and heat treatment at 90°C, 120°C and 150°C. The effect, on the films, of hardening methods, water uptake, moisture, drug release and antimicrobial activity against Staphylococcus aureus was evaluated. The O-carboxymethylchitosan films were soluble in simulated saliva and after treatment, and the water uptake of the films decreased as the temperature or presence of glutaraldehyde increased. The antimicrobial activity of tetracycline was preserved, and efficiency was dependent on the hardening treatment to which the films were submitted.