Oral health in immature permanent teeth with traumatic injuries is particularly vulnerable, and regenerative endodontic treatment (RET) using stem cells from the apical papilla (SCAP) holds potential for root development and tissue regeneration. However, bacterial persistence, especially Enterococcus faecalis, poses a challenge to successful treatment outcomes. To address this, we evaluated the probiotic Lactobacillus gasseri for its co-aggregative and anti-adhesive properties against E. faecalis. An in vitro aggregation test demonstrated effective co-aggregation between the probiotic and opportunistic strains. Additionally, flow cytometry analysis revealed that E. faecalis binding to SCAP was significantly reduced when the L. gasseri concentration was nine times higher. To substantiate these findings, an in vivo Drosophila melanogaster gut model was used, where immunofluorescence imaging and culture-based methods confirmed decreased E. faecalis adhesion at both 1:1 and 9:1 probiotic-to-opportunistic ratios. These results highlight L. gasseri B16 as a promising probiotic strain to improve RET outcomes.