Guaco Mikania glomerata Spreng. and M. laevigata Sch. Bip. ex Baker, Asteraceae, has antimicrobial activity and may be helpful in reducing the incidence of oral diseases. This double-blinded randomized clinical trial aimed to evaluate the efficacy of guaco mouthwashes on the disinfection of toothbrushes used by preschool children, tested positive for mutans streptococci (MS), as well as the quantification of its coumarin contents by high performance liquid chromatography. Ethanol extracts were obtained by percolation. The mouthwashes were prepared with 2.5% g/mL M. glomerata and M. laevigata ethanol extracts, standardized for their coumarin content (% mg/mg). Antimicrobial effect of the mouthwashes and extracts were assessed in vitro against Streptococcus mutans (ATCC 25175TM), using 2.4 to 500 µg/mL to calculate the minimum inhibitory concentration (MIC). For the in vivo study, 24 patients were randomly assigned to a 4-stage changeover system with a one-week interval between each stage. All solutions were used in all stages by a different group of children. After brushing without toothpaste, toothbrushes (n=96) were sprayed with water and solutions of M. glomerata (2.5%), M. laevigata (2.5%) and chlorhexidine (0.12%). Microbiological analysis was carried out after 4 h and 30 days, respectively. MIC values were 400, 125 and 14 µg/mL, respectively, for both crude ethanol extracts, mouthwashes of M. glomerata and M. laevigata. Statistical analysis showed that all solutions decreased contamination of toothbrushes by mutans streptococci (chlorhexidine 50.7±17.7%; M. glomerata 37.3±23.7% and M. laevigata 28.7±25.1% of inhibition). Treatment with chlorhexidine and M. glomerata were statistically similar (p>0.05). M. glomerata mouthwash could be useful in herbal strategy programs against mutans streptococci and the marker coumarin may be not related to the activity observed.