Objective: The Er:YAG laser with photon-induced photoacoustic streaming (PIPS) technique was reported to be effective in root canal disinfection. This study attempted to further investigate the antibacterial efficacy and smear layer removal ability of PIPS in comparison with conventional syringe irrigation in vitro. Methods: For antibacterial analysis, 48 single-rooted human teeth were prepared and inoculated with Enterococcus faecalis, and then divided into six groups of eight roots each. The colony-forming units (CFUs) per milliliter were determined after infection as the baseline. Then, the teeth were subjected to either PIPS plus 3% sodium hypochlorite (PIPS + NaOCl) or conventional syringe irrigation with 0.9% saline, 3% NaOCl, 17% ethylenediaminetetraacetic acid (EDTA), 0.2% chlorhexidine gluconate (CHX), or 3% NaOCl alternating with 17% EDTA. The reduction of CFUs in the individual group was determined. Additionally, scanning electron microscopy (SEM) examination of the canal walls for E. faecalis colonization was performed. For comparing the smear removal efficacy, another 48 single-rooted teeth, assigned to different groups as mentioned, were irrigated after mechanical instrumentation. The presence of a smear layer at different levels of the root canal was scored by SEM examination. Results: No significant differences were found in CFU reduction. No bacteria could be observed by SEM in the NaOCl, NaOCl + EDTA, and PIPS + NaOCl groups. The scores of smear layer of the NaOCl + EDTA and PIPS + NaOCl groups were significantly lower than those of the other groups in the coronal and middle third of the root canal. None of the methods can effectively remove smear layer in the apical third. Conclusions: PIPS system supplied with NaOCl and conventional syringe irrigation with NaOCl + EDTA are comparable in their ability to remove E. faecalis and smear layer in single-rooted canals.