Endodontic infections are polymicrobial diseases characterised by complex interactions between diverse microbial communities and the host immune response. Advances in molecular microbiology have substantially enhanced the understanding of the microbial ecology of infected root canals, demonstrating that these infections are dominated by structured biofilm communities rather than isolated planktonic microorganisms. The persistence of endodontic infections is attributed to intricate microbial interactions, biofilm associated antimicrobial tolerance, virulence factor expression, and sophisticated immune evasion mechanisms. Conventional culture-based techniques have provided valuable insights into endodontic pathogens, however, culture independent approaches, including polymerase chain reaction, fluorescence in situ hybridisation, metagenomics, and next generation sequencing, have considerably expanded the identification of previously undetected microorganisms and their functional characteristics. Emerging evidence also highlights the contribution of fungi, viruses, and archaea to persistent infections, further emphasising the complexity of the endodontic microbiome. In parallel, increasing antimicrobial resistance among endodontic pathogens presents a significant therapeutic challenge and necessitates the development of novel disinfection strategies and precision based therapeutic approaches. This review summarises the current understanding of the microbial ecology, pathogenic mechanisms, diagnostic advances, and future perspectives in endodontic microbiology, providing an evidence-based overview of their implications for contemporary endodontic practice.