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Ultrafiltration is widely used to treat various environmental waters, and on-line membrane cleaning with various chemical reagents is frequently employed to sustain the filtration flux. However, the residue of cleaning agents in the ultrafiltration system is unavoidable, which may affect microbiological properties and biofilm formation during the next-round filtration. By investigating the changes in microbial characteristics, and their biofouling behaviors after exposure to HCl, NaOH, NaClO, citric acid (CA), and sodium dodecyl sulfonate (SDS), this study fills a knowledge gap in microbial responses to various types of chemical cleaning agents in an ultrafiltration system. The result shows that HCl, NaOH, and NaClO affect the bacterial properties and subsequent attachment on the membrane surface, while CA and SDS have no obvious influence on microorganisms. Specifically, HCl, NaOH, and NaClO reduce the hydrophobicity and mean size of suspended microorganisms, increase the extracellular polymeric substances (EPS) release, and trigger intracellular reactive oxygen species (ROS) generation, resulting in the death of a large quantity of microorganisms. Due to the self-protecting strategy, plenty of living cells aggregate on the membrane surface and form a cake layer with a stratified structure, causing more severe membrane biofouling.