Aim Adherence of bacteria to dentine is a prerequisite to infection of the root canal system, yet adherence of root canal bacteria to dentine is poorly understood. The aim of this study was to evaluate the effect of conditioning films and anti‐adherent compounds on bacterial adherence to dentine. Methodology Freshly extracted molar teeth were prepared and sectioned to give 225 discs of predetermined dimensions. The discs were allocated to two groups. Group 1 (n = 189) was divided into three subgroups (n = 63) and coated with one of three conditioning agents (artificial saliva, serum, or distilled water) prior to bacterial inoculation. Group 2 discs (n = 36) were treated with either a novel anti‐adherent agent (PC1036, Biocompatibles) (n = 18) or distilled water (n = 18) prior to conditioning with artificial saliva. Monospecies bacterial biofilms were generated on the dentine discs by incubating them in brain heart infusion broth (37 gL−1) containing Streptococcus intermedius (Si), Enterococcus faecalis (Ef) or Lactobacillus fermentum (Lf) (originally isolated from infected root canals). The number of bacteria adhering to the discs in each of the groups was determined using standard serial dilution protocols. Additional discs were prepared under all conditions for scanning electron microscopy. Where appropriate, statistical analysis by one way anova, post hoc Bonferroni, and independent t‐test were used. Results Si adhered significantly better to dentine when conditioned with serum compared with artificial saliva (P = 0.005) or distilled water (P = 0.009). Conversely, Ef adhered significantly better to the control discs (distilled water) compared with serum conditioned discs (P = 0.016). The conditioning films had no effect on the adherence of Lf, which adhered to the dentine discs significantly less (P = 0.001) than either Si or Ef. The anti‐adherent coating significantly reduced the number of Si adhering to the dentine compared with the control (P = 0.012). Conclusion Given the importance of adherence in root canal infection it is conceivable that an anti‐adherent compound, could be used to prevent bacterial recontamination of cavities or the root canal system.