Objectives To evaluate the microbial contamination of pacifiers by Mutans Streptococci (MS) and the efficacy of different methods for their disinfection. Methods Twenty-eight children were assigned to a 4-stage changeover system with a 1-week interval. In each stage, children received a new pacifier and the parents were instructed to maintain their normal habits for 1 week. After this time, the pacifiers were subjected to the following 4 disinfection methods: spraying with 0.12% chlorhexidine solution, Brushtox® or sterile tap water, and immersion in boiling tap water for 15 minutes. Microbiological culture for MS and Scanning Electron Microscopy (SEM) were performed. The results were analyzed statistically by Friedman’s non-parametric test (a=0.05). Results The 0.12% chlorhexidine spray was statistically similar to the boiling water (p>0.05) and more effective than the Brushtox® spray and control (p