Protium heptaphyllum (PH) is rich in essential oil, has anti-inflammatory properties and has no toxic potential. However, data is unavailable about its antiseptic effect against bacteria that cause caries. This study aimed to evaluate the antiseptic effect of the toothbrush rinse of essential oil of PH as well as its chemical stability. The toothbrush rinse was prepared with 1% essential oil of PH. The minimum inhibitory concentration (MIC) of the essential oil and toothbrush rinse were evaluated against Streptococcus mutans (ATCC 25175™). The ex vivo study was double-blinded and randomised; the children were divided into three groups, each participating in a crossover design where all solutions (water, toothbrush rinse (1%) and chlorhexidine (0.12%) were used in all stages by different groups of children. The chemical composition of the essential oil and toothbrush rinse were analysed by gas chromatography coupled to mass spectrometry. The stability was evaluated at three time points. The essential oil and toothbrush rinse exhibited antimicrobial activity against S. mutans, MIC = 0.125 and 2.4 µg/ml. The toothbrush rinse showed the same effect as chlorhexidine on disinfecting the toothbrushes contaminated with mutans streptococci (pH = 57.3 ± 5.3%; chlorhexidine 55.5 ± 13.3%; water 39.4 ± 5.8%; p > 0.05). Chromatographic analysis showed that the essential oil contained monoterpenes as a major component, and the toothbrush rinse possessed the same constituents as the pure essential oil, except for α-terpineol. Storage did not cause chemical degradation of the toothbrush rinse, but decreased the concentration of the chemical constituents. The toothbrush rinse of essential oil of P. heptaphyllum showed antiseptic properties and exhibited antimicrobial activity against mutans streptococci. Key words: Mutans streptococci, Protium heptaphyllum, antimicrobial, monoterpenes, chemical composition, contamination of toothbrushes.