Background <p>Removing infectious bacteria biofilms from the root canal system is crucial for a successful endodontic treatment. This study investigated the antibacterial effect of femtosecond laser (fs-laser) against <i>Enterococcus faecalis</i> (<i>E. faecalis</i>) and <i>Fusobacterium nucleatum</i> (<i>F. nucleatum</i>) biofilms on dentin.</p> Methods <p>The chemical composition of dentin slices from extracted human teeth was analyzed using FTIR and Raman probes. The morphology of fs-laser ablated dentin grooves was evaluated by an optical profiler, and the fs-laser ablation fluence threshold was obtained by a mathematical model. A correlation between dentin chemical composition and ablation threshold was established. The antibacterial effect of different fs-laser irradiation dosages within the safe threshold on <i>E. faecalis</i> and <i>F. nucleatum</i> biofilms was firstly evaluated using the growth curve method. The biofilm removal efficacy on dentin and antimicrobial effect in dentinal tubules was further evaluated by CLSM and SEM analysis. The effect of fs-laser irradiation on the microhardness of dentin surface was also evaluated. The fs-laser irradiation process was observed using a spectrometer.</p> Results <p>The peak intensity of phosphate group showed a positive correlation to the fs-laser dentin ablation fluence threshold in both FTIR and Raman spectroscopy. The safe fluence threshold of 1.8&#xa0;J/cm² was determined by a prediction model on 20 dentin samples. The antimicrobial effect of fs-laser increased along with the irradiation fluence or time. Both <i>E. faecalis</i> and <i>F. nucleatum</i> biofilms on dentin could be effectively removed by the fs-laser with 1.5&#xa0;J/cm² fluence for 20&#xa0;s without compromising the microhardness of dentin surface. Meanwhile, fs-laser could also eliminate the bacteria in dentinal tubules. The generation of plasma occurred during the fs-laser irradiation process, and the plasma spectra exhibited distinguishable characteristics between the two kinds of biofilms.</p> Conclusions <p>Fs-laser could effectively remove both <i>E. faecalis</i> and <i>F. nucleatum</i> biofilms on dentin, along with a notable antibacterial effect in dentinal tubules.</p>