On-line membrane cleaning with NaClO is frequently implemented during membrane-based water treatment, where diverse aqueous microorganisms inevitably react with NaClO, leading to a significant impact on the next-round operation. This study filled the knowledge gap to explore the respective response of bacteria, algae and fungi to NaClO exposure. The results indicated that the three types of microorganisms exhibited distinct behaviors to NaClO attack. Specifically, E. coli showed stronger adaptation to NaClO by releasing more EPS and agglomeration behavior to resist chemical attack, thereby maintaining a higher survival rate. In contrast, M. aeruginosa and Aspergillus might experience significant cell death caused by substantial production of ROS. During subsequent ultrafiltration, accelerated biofouling development with a thinner biofilm layer was found for all three microorganisms exposed to NaClO, especially for M. aeruginosa and Aspergillus who formed the biofilms with remarkable stratified structures. The biofilm formed by E. coli was strongly correlated with EPS secretion, while excessive production of ROS and attachment of survived cells on membrane were considered as crucial factors controlling the biofilm development for M. aeruginosa and Aspergillus. Detailed responding mechanisms to NaClO cleaning were eventually proposed for the respective three microorganisms, which offered new insight into UF system operation for water treatment.