This study investigated the efficacy of Er,Cr:YSGG laser and ultrasonic activated irrigation on eradicating a mixed-species biofilm grown in root canals with complex anatomy. The biofilm was grown over 4-weeks in the root canals of decoronated human mandibular molar teeth. Control roots received no further treatment. The remaining roots were chemomechanically prepared using different irrigating protocols: 4% NaOCl and 15% EDTAC with ultrasonic activated irrigation and laser activated irrigation using power settings of 0.5 W and 0.75 W. Cellular viability was determined using serial plating. One tooth from each group was subjected to qualitative SEM analysis. Quantification by culturing revealed significant differences between control group and all other treatment groups. This study demonstrated that chemomechanical irrigation with laser and ultrasonic activated irrigation significantly reduced the bacterial load from complex root canal systems; however, there were no significant differences found between the experimental groups.