The need for controlling bacteria and pain during root canal therapy is undeniable. This clinical trial aimed to assess whether there is a difference in colony-forming unit (CFU) reduction after instrumentation and post-endodontic pain after root canal treatment (RCT) using a traditional endodontic cavity (TEC) versus a conservative endodontic cavity (CEC). This clinical study was conducted on 89 patients designated for a single-visit RCT. Patients were allocated randomly (TEC n = 45 and CEC n = 44). The access opening was gained accordingly in each group by a single operator. A pre-instrumentation sample of root canal dentin was collected using an endodontic file; the second sample was collected similarly, right after shaping and cleaning the root canal. The CFU was calculated based on the samples collected. The pain level was recorded preoperatively and at 1, 7, and 21 days postoperatively utilizing a visual analog scale (VAS). There were no statistically significant differences in the CFU reduction between the TEC and CEC groups (p > 0.05). Additionally, there were no statistically significant differences found in postoperative pain levels between the TEC and CEC at 1, 7, and 21 days (p > 0.05). Despite the limitations of this study, both the CEC and TEC demonstrate a decrease in bacteria within the root canals and alleviate postoperative pain with no difference between them.