Membrane fouling is a main bottleneck that limits the stable operation of membrane bioreactors (MBRs). However, the nature of residual foulants over frequent chemical cleaning remain elusive. Herein, we collected fouled membranes from a large-scale MBR with the treatment capacity of 200, 000 m³/d and explored the culprit for the key components resistant to chemically enhanced backwash. During 155 days of operation, transmembrane pressure increasing highly linked to the characteristics of membranes and bio-cakes. Fourier-transform infrared microscopy deciphered that a part of polyvinylpyrrolidone in the structure of polyvinylidene fluoride hollow fiber membrane was removed by NaOCl after chemical backwash. The stubborn uronic acids were mostly responsible for irreversible fouling due to pore constriction and the resistance to NaOCl. The fouling-related genera f_Rhodocyclaceae and f_Anaerolineaceae prevailed in the bio-cakes. According to the network analysis, the negative and competitive interactions on the bio-cake contributed more to severe membrane fouling than the positive and cooperative interactions. The keystone taxa c_WS6 (Dojkabacteria) and c_Deltaproteobacteria, which involved in secreting polysaccharides and hydrolyzing refractory matters, accelerated the accumulation of organic foulants. Our findings help to elucidate the key contributors to membrane fouling and anti-cleaning in full-scale MBR, which provide new insights for future fouling control.