Membrane biofouling, which severely limits the membrane technology application, can be mitigated by quorum sensing inhibitors (QSIs) that suppress quorum sensing (QS) and biofilm formation-related genes. However, antibiotics can potentially interfere QS pathway, thereby altering microbial community structure and biofilm formation. This study investigates the mechanism of antibiotics interference with QSIs of vanillin and methyl anthranilate on microbial gene expression along with bacterial community structure and metabolism in real surface water (SW) and secondary effluent (SE) systems during nanofiltration biofouling. We demonstrated that sulfamethoxazole (SMX) attenuated the quorum quenching (QQ) effect of vanillin by reducing its inhibitory impact on the expression of pseudomonas quinolone signal (PQS) biosynthesis genes (pqsA and pqsC), N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL) and N-butyryl homoserine lactone (C4-HSL) receptor genes (lasR and rhlR), and rhamnolipids synthesis gene (rhlA) by over 44%. SMX weakened the QQ effect of methyl anthranilate by reducing its inhibition of PQS biosynthesis genes (pqsABCDE) and 3OC12-HSL synthesis gene (lasI) by 12–62%. The coexistence of antibiotics and QSIs led to an overexpression of antibiotic resistance genes (oprM and mexAB) up to 9 times compared to antibiotics alone. Additionally, SMX and tetracycline (TET) also reduced the inhibitory effect of QSIs on dominant genera with high metabolic and secretory performance (Acinetobacter and unclassified_f_Enterobacteriaceae) and carbohydrate/amino acid metabolism genes in SW and SE systems. These findings reveal that antibiotics can interfere with QS regulatory pathways and weaken the effect of QSIs, which provides new insights into applying QSIs for membrane biofouling control in the presence of antibiotics.