The validity and biocompatibility of irrigating agents are imperative for the success of root canal therapy. The imperfections in the currently available irrigants highlight the fact that more advanced technologies and strategies are required for complete disinfection in endodontic treatments. In the present study, a Fenton reaction-enhanced antimicrobial sonodynamic therapy (SDT) platform was fabricated for root canal disinfection. Firstly, mesoporous silica nanoparticles (MSNs) were synthesized, grafted with an amino group and then conjugated with sonosensitizer protoporphyrin IX (PpIX). Iron ions were then anchored (M@P-Fe) to initiate a Fenton reaction. Nanoparticle characterization by size and zeta potential measurements, scanning electron microscopy, transmission electron microscopy and thermogravimetric analysis confirmed that the platform was successfully developed. Reactive oxygen species (ROS) generation assessment, methylene blue degradation and electron spin resonance assays illustrated upon ultrasound (US) irradiation, that augmented ROS, can be produced by US activated PpIX and iron mediated Fenton reactions from low concentration H2O2 (0.01%). In vitro anti-Enterococcus faecalis efficacy was demonstrated by growth curve and colony forming unit measurements. Confocal laser scanning microscopy and scanning electron microscopy observations illustrated the effectiveness of the platform on in situ biofilm eradication in root canal. Owing to the stronger oxidizing capability and short lifetime of ROS, the Fenton reaction-enhanced SDT can induce detrimental oxidative damage to bacteria upon activation of US while avoiding nonspecific toxicity to cells, which was verified by cytotoxicity evaluations using CCK-8 assay and morphology observation of MC3T3-E1 cells. Compared to commonly used NaClO, this nanoplatform displayed desirable anti-bacterial, anti-biofilm abilities and better biocompatibility. These results highlight that the integrated M@P-Fe + US + H2O2 platform is a promising candidate for US enhanced root canal irrigation and disinfection.