The purpose of the present study was to evaluate the effect of Revo-S and ultrasonic instrumentation with different irrigations on dentinal surface and bacterial contents of root canals. Sixty four recently extracted sound human maxillary molar teeth were divided into two groups ; 32 roots each, Group I:Root canals were instrumented with the rotary system (Revo-S). Group II:Root canals were instrumented with the ultrasonic system. Each group was subdivided into 4 subgroups of 8 roots each as follow: Group I: divided into : Subgroup IA: irrigated with 2 mL EDTA. Subgroup IB: irrigated with 2 mL Naocl. Subgroup IC: irrigated with 2 mL Chlorhexidine. Subgroup ID: irrigated with 2mL distilled water. Group II: Divided into: Subgroup IIA: irrigated with 2 mL EDTA. Subgroup IIB: irrigated with 2 mL Naocl. Subgroup IIC: irrigated with 2mL Chlorhexidine Subgroup IID: irrigated with 2mL distilled water. A classical bacterial counting technique was used for each group. The cleanliness of each root (smear layer) was evaluated by SEM at three areas by means of a numerical evaluation scale. Scoring system consisted of 5 criteria. Measuring the area of dentin debris was done using the digital image analysis. The results obtained from this study indicated that the Revo-S prepared groups showed higher percentage of reduction of the bacterial counts than their counter specimens used in the ultrasonic prepared groups. The groups prepared with Revo-S showed lower mean and standard deviation values of remaining amount of the smear layer and debris than the groups prepared with ultrasonic system. The least mean and standard deviation values for the amounts of remaining smear layer and debris were obtained when EDTA was used. NaOcl showed less mean and standard deviation values of the amount of smear layer and debris than the CHX which also showed less mean and standard deviation values for the amount of remaining smear layer and debris than distilled water.