Introduction Methods of decontamination or sanitization of toothbrushes have been questioned. Objective This study assessed the effectiveness of pomegranate peels infusion as a disinfectant of toothbrushes against Streptococcus mutans. Material and method A sample of 16 schoolchildren aged between 7 and 9 years performed brushing 5 days/week, with a careful brushing once a day. After each day of brushing, the toothbrushes were washed and sprayed with one disinfectant solution. This procedure was repeated for 4 weeks using one of the different solutions per week: distilled water (G1; negative control), pomegranate (Punica granatum Linn) peels infusion (G2), 1% sodium hypochlorite (G3) and 0.12% chlorhexidine digluconate (G4). After the fifth day, toothbrushes were collected for laboratory analysis. Toothbrushes heads were subjected to agitation in saline dilution of 10–1, 10–2,10–3, and 25 μL of each dilution were seeded in mitis salivarius agar culture medium for S. mutans colony-forming unit (CFU) counting. One calibrated examiner (Kappa = 0.91) performed the CFU (mL–1 × 104) counts. Kruskal-Wallis and Dunn Multiple Comparison tests were used at a significance level of 5%. Result G1 presented the highest number of CFU (3.9 ± 8.4), followed by G2 (3.2 ± 4.0). No S. mutans growth was observed in G3 and G4. There was no statistically significant difference between G1 and G2 and between G3 and G4 (p>0.05). Conclusion Pomegranate infusion was completely ineffective for the disinfection of toothbrushes against S. mutans when compared with 1% sodium hypochlorite and 0.12% chlorhexidine digluconate solutions.