The aim of the study was to evaluate toothbrush contamination in vivo by Candida spp. and the efficacy of Periogard and Neem Sattiva, in spray, in the disinfection of these toothbrushes. This study was performed in three phases in which mouthrinses and sterile distilled water (control group) were sprayed six times on toothbrush bristles used by 61 university students. Toothbrushes were then submitted to microbiological processing for the isolation and identification of Candida species. Fifty-nine students completed the three phases of this study, and 22 (37.3%) control group toothbrushes presented growth of Candida species. Periogard and Neem Sattiva eliminated growth of Candida spp. in 48.1 and 7.4% of toothbrushes, respectively. Contamination by Candida spp. was observed on various toothbrushes of the control group. Periogard was more efficacious than Neem Sattiva in eliminating growth of Candida spp. on the toothbrush bristles.