The objectives of this study were to compare the effects of various acid solutions combined with ultrasonics as an aid to remove mineral trioxide aggregate (MTA)-based root canal filling and to assess their effect on the surface topography and microhardness of root canal dentin. Materials and Method: Fifty human permanent single rooted and single canaled freshly extracted teeth were decoronated and sectioned apically to prepare the middle third of root sections of 5 mm length. The canals were prepared in a step-back manner. OrthoMTA was packed throughout the prepared canals. These root sections were incubated for one week and subsequently randomly allocated to five groups (n = 10) according to the OrthoMTA removal method: No treatment (NT); 5% glycolic acid + ultrasonics (5% GA+U); 10% glycolic acid + ultrasonics (10% GA+U); 10% citric acid + ultrasonics (10% CA+U); Distilled water + ultrasonics (DW+U). A 1 mm deep well was created within the coronal end of the set OrthoMTA. Wells were filled with each respective test solution and left for 5 min. Thereafter, further removal of OrthoMTA used a specific ultrasonic tip. Finally, the canals were flushed using 1 mL of the respective test solutions and activated with a Controlled Memory ultrasonic tip for two cycles of 20 s each followed by flushing with 1 mL of distilled water and paper point drying of the canals. Then, specimens were longitudinally split into two halves and examined under a scanning electron microscope (1000×) to assess the residual OrthoMTA and surface topography of root canal dentin. The Vickers surface microhardness of treated radicular dentin was measured using the HMV-2 microhardness tester. Result: Data were analysed using one-way ANOVA followed by Tukey’s post hoc test. Significant differences for residual OrthoMTA were observed between (10% GA+U) with (5% GA+U), (10% CA+U), (DW+U) and (NT) (pvalue < 0.01). In the context of microhardness, (5% GA+U) and (10% GA+U) showed statistically significant difference compared to (NT), (10% CA+U) and (DW+U) (pvalue < 0.01). Conclusion: 10% GA+U was superior to other tested groups in removing OrthoMTA, but it substantially reduced dentin microhardness.