INTRODUCTION: This study aimed to assess changes in the fluorescence characteristics of Enterococcus faecalis in human dentine over a period of 24 hours following treatment with endodontic irrigants. METHOD: Sterilised, non-functional extracted third molars were embedded in acrylic resin and uniformly sectioned into 2mm thick dentine sections. After the removal of smear layer, the dentine sections were inoculated with E. faecalis and cultured for 7 days. The infected dentine sections were subsequently treated with different concentrations of sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2). Bacterial fluorescence readings were assessed at different time points using a calibrated laser device. All data were assessed for normality (Kolmogorov Smirnoff test) and analysed using ANOVA with Bonferroni post-hoc tests. RESULTS: Fluorescence readings were quenched when E. faecalis infected human dentine sections were treated with oxidizing irrigants in vitro. Throughout a 24-hour period, fluorescence recovered in part but did not return to baseline level. CONCLUSION: The fluorescence quenching effect of these oxidizing agents needs to be considered when using laser fluorescence in assessing the quality of root canal debridement or disinfection.