Rapid advancements not only facilitate human adaptation but also trigger environmental adjustments. While pivotal discoveries like Penicillin revolutionized medicine, the subsequent overuse of antibiotics led to diminishing efficacy due to antibiotic resistance. Addressing biofilm formation as a major contributor to antimicrobial resistance and recognizing quorum sensing as a key factor in biofilm formation, there is a need for new strategies. Glutathione, a natural antioxidant, has shown promising potential as an effective antimicrobial agent and a reliable component for cellular defence in the immune system. This study explores the capability of Glutathione to mitigate quorum sensing-induced biofilm formation in Klebsiella pneumoniae and Serratia marcescens. The results demonstrated that glutathione induced ROS-mediated cell death in these bacteria. Glutathione exhibited a maximum inhibition of approximately 85% in biofilm formation for both K. pneumoniae and S. marcescens. It also effectively disrupted preformed biofilms by degrading the eDNA of the EPS layer of matured biofilms. Interestingly, glutathione attenuated the quorum sensing pathway, as evidenced by reduced production of virulence factors, thereby mitigating QS-induced biofilm formation in both bacteria. This work lays the groundwork for further exploration in developing glutathione as a novel antibiotic to combat antibiotic resistance.