Toothbrushes are sometimes contaminated by micro-organisms that can cause infection. This study evaluates the efficacy of Citrus hystrix sprays compared with 0.12% chlorhexidine gluconate (CHX) for disinfecting bacteria on children's toothbrushes. The participants were 61 children with a high caries risk aged 8-11. Each subject received two toothbrushes to be used continually for 7 consecutive days. The first set of toothbrushes was used as a baseline without any disinfectant. The others were divided into 5 groups and sprayed with distilled water (control), 0.12% CHX, 6% makrut oil (MO), 10% MO, or 13% MO. Toothbrushes were placed vertically into sterile test tubes containing 8 ml of TYS20B medium and incubated overnight. Then we performed 10-fold serial dilutions from 10 −1 -10 −5 , and spread them on BHI or MS agars for investigating facultative bacteria, oral streptococci, andStreptococcus mutans. Results showed that for total facultative bacteria, 10% MO and 13% MO gave the highest reduction rate (100%) > CHX (88%) > 6% MO (81%). For S. mutans, 10% MO and 13% MO also showed the highest reduction rate (100%) > 6% MO (90%) > CHX (88%). For oral streptococci, 13% MO gave the highest reduction rate (100%) > 10% MO (91%) > CHX (88%) > 6% MO (69%). There was no significant difference between oral sprays and 0.12% CHX in the reduction of total facultative bacteria, oral streptococci, and S. mutans (p > 0.05).