A bacterial strain capable of efficiently degrading pentabromobiphenyl ether (BDE-99) was isolated from activated sludge and named as NLPSJ-22. This strain was highly close to Pseudomonas asplenii with 100% similarity. The degradation products of BDE-99 were analyzed by gas chromatography mass spectrometry. The biochemical degradation pathways analysis indicated that BDE-99 gradually transformed to diphenyl ether by meta-, para- and ortho-debromination. It became phenol under the action of ring-opening cracking and finally entered the tricarboxylic acid cycle. The degradation of BDE-99 by strain NLPSJ-22 conformed to the first-order reaction kinetics. Rhamnolipid significantly improved the cell-surface hydrophobicity and the degradation of BDE-99. The highest degradation efficiency (96%) was achieved when diphenyl ether as co-metabolic substrate was added. In the bioaugmentation membrane bioreactor (MBR) system, BDE-99 was intensively degraded, and the reactor reached a steady state in about 35 days. The degradation rate of BDE-99 was over 80%, which was significantly higher than that of the control system. MiSeq sequencing results indicated that the genera of Rhodococcus, Bacillus, Pseudomonas, Burkholderia, and Sphingobium were the predominant bacterial communities responsible for BDE-99 biodegradation in the MBR. Pseudomonas increased significantly in the bioaugmented reactor with the relative abundance increasing from 5% to 24%.