Objective <p>To assess disinfectants and adjunctive techniques against Enterococcus faecalis biofilms in extracted single-rooted human teeth.</p> Methods <p>A PRISMA-guided review of PubMed, EMBASE, Cochrane, and Web of Science was conducted to identify in vitro studies utilizing extracted single-rooted teeth. Study quality was evaluated using a 13-item checklist adapted from JBI/CRIS guidance. Evidence was synthesized qualitatively and graded based on methodological limitations, relevance, consistency, and data adequacy.</p> Results <p>Ninety-seven studies met the inclusion criteria. Mechanical preparation effectively reduced bacterial load but did not completely eradicate biofilms. Conventional irrigants produced variable results; however, combined protocols and activation methods often yielded the best disinfection outcomes. Physical activation enhanced irrigant penetration and antimicrobial effects. Antimicrobial photodynamic therapy (aPDT) proved beneficial as an adjunct in certain protocols but was sensitive to specific parameters. Calcium hydroxide demonstrated limited efficacy, while triple antibiotic paste approached sterilization but raised concerns about resistance. Nanoparticle and natural irrigants showed potential but were generally less effective than sodium hypochlorite and require further validation. Only 28% of the studies were assessed as low risk of bias, with common shortcomings including inadequate blinding, inconsistent use of neutralizers, and heterogeneous outcome reporting.</p> Conclusions <p>No single method reliably eradicates Enterococcus faecalis biofilms; however, combined mechanochemical approaches with activation or aPDT appear most promising. Standardized, clinically relevant studies are necessary for further advancement.</p>