Dental caries is considered a major health burden, and preventive strategies are needed to improve oral health. It is suggested that natural essential oils possess anti-plaque formation properties and exhibit strong antimicrobial activity; however, in vivo studies to support these concepts are scarce. We evaluated the effects of tea tree oil (TTO) on caries initiation and progression in vivo to generate supportive data for clinical studies in patients at high risk of caries. We first assessed TTO in vitro against Streptococcus mutans and Streptococcus sobrinus, two of the most common oral bacteria associated with dental caries development, using bacterial growth assays, biofilm formation, and adhesion assays. TTO efficacy on caries initiation and caries lesion progression was then evaluated in vivo, where complex biofilms are formed on dental enamel. Our results showed that TTO demonstrates strong antimicrobial efficacy by inhibiting bacterial growth and biofilm formation while preventing bacterial adhesion on human teeth. In vivo, TTO application reduced the number and depth of carious lesions. Specifically, the number of caries lesions was lower in the TTO-treated group compared to the control group (13 vs. 19 lesions), and the lesion area was significantly smaller in the TTO-treated group compared to the untreated group (p = 0.003). TTO did not affect the extent of reparative dentin formation. The clinical relevance is primarily for individuals who have difficulties brushing their teeth or those at high risk of developing dental caries, serving as an adjunct to preventive dentistry.