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Introduction: Endodontic treatment failures often stem from the presence of microbial pathogens, particularly Enterococcus faecalis and Candida albicans . This study systematically assesses the prevalence of E. faecalis and C. albicans in endodontic retreatment cases, aiming to explore their impact on treatment outcomes., Methods: Employing a systematic sampling approach, 30 patients with a history of previous endodontic treatment were selected. Rigorous clinical and radiographic assessments were conducted, following standardized protocols for root canal sample collection. Microbiological analysis, utilizing selective culture media, was employed to identify and quantify E. faecalis and C. albicans. Statistical analyses, including chi-square and logistic regression tests, were performed., Results: The study involved 30 patients undergoing endodontic retreatment, with comprehensive clinical and radiographic evaluations for cases with and without periradicular lesions. Microbiological analysis unveiled a significant prevalence of E. faecalis and C. albicans, establishing a robust association between these pathogens and retreatment failure. These findings underscore the critical need for targeted antimicrobial interventions to enhance the overall success rates of endodontic retreatment procedures., Conclusion: This study highlights the substantial prevalence of E. faecalis and C. albicans in endodontic retreatment cases, emphasizing the importance of identifying and effectively managing these pathogens for successful treatment outcomes. The notable association between these microbial agents and retreatment failure underscores the imperative for tailored antimicrobial strategies to enhance the efficacy of endodontic retreatment procedures., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)