OBJECTIVE Long-term use of orthodontic appliances and fixation ligatures creates a favorable environment for the accumulation of oral normal microflora and increases the risk of enamel demineralization and periodontal disease. The aim of this study was to compare the antimicrobial effects of Zataria Multiflora extract and 0.2% chlorhexidine (CHX) mouthwash on experimentally contaminated orthodontic elastomeric ligatures. METHODS In this lab trial study, Iranian and foreign-made elastomeric ligatures were experimentally contaminated in Streptococcus mutans, Enterococcus faecalis and Candida albicans suspensions. Ligatures were then decontaminated using 0.2% CHX as the control, 0.5 mg/ml Zataria multiflora extract mouthwashes as the test and phosphate buffered saline (PBS) as the negative control for one hour. Antimicrobial properties of both solutions were evaluated by comparing the mean viable bacterial cell count on both rings after decontamination, using SPSS version 15 software. RESULTS The mean viable bacterial cell count on Iranian ligatures was greater than that on foreign-made ligatures before disinfection (P=0.001), however this difference for C. albicans was not statistically significant (P=0.061). Chlorhexidine mouthwash completely eliminated all tested microorganisms attached to both elastomeric rings, but Zataria extract was only capable of completely eliminating C. albicans from both ligatures. Statistically significant differences were found in viable bacterial counts on both ligatures before and after disinfection with Zataria extract (P=0.0001). CONCLUSIONS Zataria multiflora extract has antimicrobial properties and can be used for disinfection of elastomeric ligatures. In vivo studies are required to evaluate the efficacy of the incorporation of this herbal extract in mouthwashes for orthodontic patients.