Nonylphenol ethoxylate (NPEO)-degrading bacteria were isolated from activated sludge using an improved isolation method, and the corresponding degradation behaviours were investigated. Eight NPEO-degrading strains distributed in genera Pseudomonas, Sphingomonas, Sphingobium, Cupriavidus, Ralstonia, Achromobacter, and Staphylococcus were acquired. The latter five genera have never been reported for the degradation of NPEOs. Four degradation patts were observed for the eight pure strains. In patt A, NPEOs were converted to short-chain NPEOs and carboxylated products, while in patt B, lower ethoxylated oligomers appeared. Nonylphenol monoethoxylate was the main product in patt C, while in patt D ethoxylated units was oxidized but not shortened. Patt C and D have not yet been reported.