Prior investigations concerning quorum quenching (QQ), which hinders quorum sensing (QS) in microbial communication, concentrated predominantly on N-acylhomoserine lactones (AHLs), which are communicated merely by gram-negative bacteria. However, to combat biofouling more effectively, particularly biofilm-related issues, it is important to explore the inhibition of autoinducer-2 (AI-2), a universal signal (interspecies communication) employed by both gram-negative and gram-positive bacteria. This study aimed to isolate and identify novel strains capable of more effectively inhibiting AI-2 signaling for use in engineering fields. The results revealed that the newly isolated strain Pantoea sp. PL-1 demonstrated remarkable efficacy in attenuating AI-2 signals, leading to a substantial reduction in the AI-2 precursor (S)-4,5-dihydroxy-2,3-pentandione (DPD) in both its pellet (with a DPD removal efficiency of 90.8 % after 180 min, with a rate constant of k = 0.8 h−1) and supernatant form (66.9 %, k = 0.37 h−1). Interestingly, the findings indicate that strain DKY-1, a strain previously reported to exhibit AI-2 QQ activity, only reduced DPD levels in its supernatant form (extracellular activity), while both the pellet and the supernatant of strain PL-1 demonstrated AI-2 QQ activity, indicating both intracellular and extracellular QQ activity. Adding the PL-1 supernatant to co-cultures of two AI-2 QS strains, E. coli K12 and P. mirabilis, successfully reduced DPD by 40 % and 70 %, respectively, without impeding their growth. Additionally, when assessing the impact of PL-1 to minimize membrane biofouling in a membrane bioreactor, it demonstrated superior performance compared to the DKY-1 strain, with a 40 % improvement. The AI-2 QQ compound of PL-1 has been identified as highly hydrophilic and low-molecular-weight, and its exact properties have not yet been fully elucidated. Therefore, uncovering its identity may be an important goal for future research.