This study evaluated the bacterial disinfection efficacy of three different irrigation solutions and their combined usage in dentin tubules contaminated with mixed biofilms. A total of 60 single-rooted mandibular premolars were prepared with ProTaper Next X4, sterilized, and then inoculated with mixed biofilm for 7 days. After biofilm formation, the teeth were randomly divided into six groups (n = 10) based on the irrigation solution used: Group 1: sodium hypochlorite (NaOCl), Group 2: chlorhexidine gluconate (CHX), Group 3: polyhexamethylene biguanide (PHMB), Group 4: NaOCl + CHX, Group 5: NaOCl + PHMB, Group 6: distilled water (control group). The teeth were sectioned at the coronal, middle, and apical thirds and stained with LIVE/DEAD BackLight and examined under a confocal laser scanning microscope (CLSM) to determine the depth of dead bacterial penetration into the dentinal tubules. Additionally, one tooth from each group was subjected to bacterial viable counting before and after irrigation. The CLSM analysis revealed that the greatest penetration of dead bacteria occurred in the coronal third, followed by the apical and middle third (p < 0.05). Combined solutions demonstrated significantly higher disinfection efficacy compared with PHMB alone (p < 0.001). Viable count analysis revealed that Group 4 and Group 5 exhibited the greatest reduction in bacterial proliferation, respectively. The combined use of solutions was found to be more effective than the sole use of solutions. Therefore, it is recommended to combine irrigation solutions during final irrigation in clinical settings.