The continuous growth of biofilm infections and their resilience to conventional cleaning methods and antimicrobial agents pose a worldwide challenge across diverse sectors. This persistent medical, industrial, and environmental issue contributes to treatment challenges and chronic diseases. Lactic acid bacteria have garnered global attention for their substantial antimicrobial effects against pathogens and established beneficial roles. Notably, their biofilms are also predicted to show a promising control strategy against pathogenic biofilm formation. The prevalence of biofilm-related problems underscores the need for extensive research and innovative solutions to tackle this global challenge. This novel study investigates the effect of different extracts (external, internal, and mixed extracts) obtained from Lactobacillus rhamnosus GG biofilm on pathogenic-formed biofilms. Subsequently, external extracts presented an important eradication effectiveness. Furthermore, a 6-fold concentration of these extracts led to eradication percentages of 57%, 67%, and 76% for Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa biofilms, respectively, and around 99.9% bactericidal effect of biofilm cells was observed for the three strains. The results of this research could mark a significant breakthrough in the field of anti-biofilm and antimicrobial strategies. Further studies and molecular research will be necessary to detect the molecules secreted by the biofilm, and their mechanisms of action engaged in new anti-biofilm strategies.