OBJECTIVE To compare the effect of a combination of 20% citric acid solution and photo-activated disinfection with the use of 20% citric acid and 2.25% sodium hypochlorite solutions on bacterial load on the dentine walls in prepared canals in vivo. METHODS Sixty-four randomly selected cases were evaluated and allocated to one of two groups. In Group 1, after gaining access to the root canal, bacterial load on the canal walls was sampled using endodontic files. A further sample was taken after apex location and initial widening of the canal had been completed and the photo-activated disinfection process carried out. A final sample was taken after completion of the canal preparation using citric acid and sodium hypochlorite solutions. In Group 2, the initial sample was taken as described previously. A second sample was taken after conventional preparation using 20% citric acid and sodium hypochlorite solutions as co-irrigants. A final sample was then taken after a subsequent PAD treatment. All samples were cultured for facultative anaerobic bacteria. RESULTS Of the canals treated in Group 1 only two of the 23 canals infected showed culturable bacteria after the use of citric acid and photo-activated disinfection. Of these two canals, one was free of culturable bacteria on completion of conventional treatment but the other still contained culturable bacteria. In Group 2, four canals of the 23 infected initially, remained contaminated after conventional treatment. After subsequent photo-activated disinfection three of these four canals were free of culturable bacteria. CONCLUSIONS Results indicate that the use of a chelating agent acting as a cleaner and disrupter of the biofilm and photo-activated disinfection to kill bacteria is an effective alternative to the use of hypochlorite as a root canal cleaning system.