Objectives: The purpose of this study was to compare the effectiveness of three different instruments on cement loss, porosity and micro-crack formation, which was not evaluated before, following scaling and root planning (SRP) using micro-computed tomography (micro-CT). Methods: In this experimental study, 30 single-rooted extracted human teeth were used and divided into three groups. All the teeth were scanned with micro-CT before and after SRP. Group 1: SRP was performed with Gracey curettes, Group 2: SRP was performed by using an ultrasonic device, and Group 3: SRP was performed by using diamond burs. Cement loss from the root surface, porosity, and micro-crack formation in the root dentine were analysed. Micro-CT is used for qualitative and quantitative analysis of samples. The obtained data were analysed statistically (p < 0.05). Results: Minimum cement loss following SRP was detected with ultrasonic scaler (26.98 mm3), whereas the highest was created by diamond burs (96.20 mm3) (p < 0.05). The total porosity values after SRP were 0.278%, 0.334% and 0.252% for Groups 1, 2 and 3, respectively. Although Group 3 had the least porosity values, there was no statistically significant difference between the groups. The highest micro-crack formation was seen in Group 2 and the lowest was in Group 1 with a significant difference (p < 0.05). Conclusions: More cement loss was observed with diamond burs. Ultrasonic devices appear to be a viable alternative to instrumentation with curettes. However, ultrasonic devices should be used carefully because of micro-crack formation since the micro-crack resulting from instrumentation with hand instruments is the least of all.