Background: A triple antibiotic mixture (ciprofloxacin; CF, metronidazole; MN, and minocycline; MC) has been used for dental root canal medicaments in pulp regeneration therapy. However, tooth discolorations, cervical root fractures, and inadequate pulp-dentin formation have been reported due to the triple antibiotic regimen. Therefore, an antibiotic encapsulated biomimetic nanomatrix gel was developed to minimize the clinical limitations and maximize a natural healing process in root canal infections. In this study, minimal bacterial concentrations (MBC) of the selected antibiotics (CF and MN) were tested in 14 representative endodontic bacterial species. Then MBC of each CF and MN were separately encapsulated within the injectable self-assembled biomimetic nanomatrix gel to evaluate antibacterial level on Enterococcus faecalis and Treponema denticola. Results: Antibiotic concentrations lower than 0.2 μg/mL of CF and MN demonstrated antibacterial activity on the 14 endodontic species. Furthermore, 6 different concentrations of CF and MN separately encapsulated with the injectable self-assembled biomimetic nanomatrix gel demonstrated antibacterial activity on Enterococcus faecalis and Treponema denticola at the lowest tested concentration of 0.0625 μg/mL. Conclusions: These results suggest that each CF and MN encapsulated within the injectable self-assembled biomimetic nanomatrix gel demonstrated antibacterial effects, which could be effective for the root canal disinfection while eliminating MC. In the long term, the antibiotic encapsulated injectable self-assembled biomimetic nanomatrix gel can provide a multifunctional antibiotic delivery method with potential root regeneration. Further studies are currently underway to evaluate the effects of combined CF and MN encapsulated within the injectable self-assembled biomimetic nanomatrix gel on clinical samples.