High intensity focused ultrasound (HIFU) is used to removal Enterococcus faecalis (E. faecalis) in planktonic suspension and dental biofilm. The bacteria E. faecalis is commonly found in secondary dental infection after root canal treatment. Sealed petri dish with E. faecalis planktonic suspension is placed at the focal region of the bowl-shaped HIFU transducer of 250 kHz in a water bath. It is subjected to sonification of 30 to 120 s. It is found that the HIFU successfully lysed and removed the bacteria from counting its colony forming units (CFU), performing scanning electron microscopy (SEM) and confocal microscopy. Also, E. faecalis biofilms in human teeth are subjected to the same HIFU treatment. Similar analysis is performed with SEM and confocal microscopy. It is found that after 60 s of sonification, most of the biofilm is either removed or lysed. In conclusion, this study highlights the potential of using HIFU as non-destructive dental root canal disinfection treatment.