The aim of this study was to estimate the independent effects of biofilm accumulation and eruption stage on the occurrence of active caries lesions on occlusal surfaces of permanent molars. The sample consisted of 298 schoolchildren (6–15 years) who were examined by a calibrated examiner at a dental unit, using artificial light, a dental mirror and a WHO probe. The occurrence of visible biofilm on occlusal surfaces and the eruption stage of each permanent molar were recorded. After professional prophylaxis and air drying, the occlusal surfaces were classified as sound, caries-inactive or caries-active. To evaluate the association of eruption stage and biofilm accumulation with active caries lesions, a logistic regression model was used. Since data were clustered, odds ratios were obtained using generalized estimating equations with a logistic link function. 1,779 permanent molars were examined. All eruption stages were associated with active caries lesions. After adjustment for biofilm accumulation and type of molar, molars with occlusal surfaces partially exposed to the oral cavity were 63.6 times more susceptible to caries activity than molars with full occlusion (95% CI = 22.0–183.7). After adjustment for eruption stage and type of molar, teeth with a high degree of biofilm accumulation were 14.5 times more susceptible to caries activity than those without visible biofilm accumulation (95% CI = 6.5–32.4). No association between active caries and hardly detectable biofilm was found in this population. The present study found that the eruption stage of permanent molars is strongly associated with active caries lesions, adjusted for biofilm accumulation and type of molar.