Purpose: To assess the effectiveness of tooth wipes in removing dental biofilm from babies' anterior teeth, as well as to evaluate the babies' behaviour and the guardians' preference concerning hygiene methods. Materials and Methods: In this random blind cross-over study, 50 high caries risk babies, from 8 to 15 months old, were divided into two groups: babies with oral hygiene performed by caregivers (n = 25) or by their mothers (n = 25). The caregivers and mothers removed biofilm using three methods of oral hygiene (tooth wipes, toothbrushes and gauze), one in each experimental phase. Professional cleaning was done before each phase, which had 2 days of biofilm accumulation and 1 experimental day, when caregivers and mothers used one method to remove biofilm. Examiners blinded to the study design assessed the biofilm index at baseline, prior to and following biofilm removal using each method. The babies' behaviour and the mothers'/caregivers' preference were assessed. Results: The tooth wipes, toothbrushes and gauze significantly reduced the amount of biofilm (P 0.001). The mothers' group removed more biofilm than the caregivers' group, using toothbrushes or tooth wipes (P 0.05). Babies in the mothers' group had better behaviour using tooth wipes than toothbrushes (P 0.05). Mothers and caregivers preferred to use tooth wipes. Conclusions: Tooth wipes are effective in removing biofilm from babies' anterior teeth and are the method best accepted by mothers, caregivers and babies. Keywords: babies, dental biofilm, hygiene methods