Purpose: The objectives of this study were 1) to compare the efficacy of biofilm removal with conventional (Bitufo 22) and triple-headed (DenTrust) toothbrushes on smooth and occlusal surfaces, and 2) to verify the influence of the person who performs the toothbrushing (mother vs dentist). Materials and Methods: Twenty children aged 4 years old with sound, complete primary dentition participated in this crossover study. The quantity of biofilm was evaluated using the bacterial plaque revealer Plaque Test (Vivadent) before and after toothbrushing by the mother or dentist for 1 minute per arch. This was done at two separate appointments, one week apart, with one type of toothbrush at the first and the other type at the second appointment. The Green & Vermillion index (1960) was used for smooth surfaces and Rodrigues et al (1999) indexes for the occlusal surfaces. Data were submitted to analysis of variance. Results: A statistically significant difference was observed on biofilm removal on occlusal and smooth surfaces, regardless of the toothbrush used or who performed the brushing (P 0.0001). The triple-headed toothbrush showed a better performance than the conventional one on smooth and occlusal surfaces when the mother had brushed the teeth. The conventional toothbrush showed a better performance only on occlusal surfaces when the dentist performed the brushing (P 0.0001). The dentist removed significantly more biofilm than the mother, 76% and 53%, respectively (P 0.0001). Conclusion: It was concluded that effective biofilm removal was achieved with both toothbrushes; however, the tripleheaded type had a better performance on surfaces when the mother brushed the teeth. The dentist removed more biofilm than the mother. Keywords: toothbrushes, effectiveness, oral hygiene, dental plaque, preschool children