[Background] Chlorinated benzenes (CBs) are widely used for the production of many important industrial compounds. High toxicity and environmental persistence of CBs bring serious threats to human health and the environment. Developing new methods for efficiently degrading CBs has become hot topics in this field. [Objective] The objective of this study was to construct a novel synthetic system, containing nanoscale zero-valent iron (NZVI) and Pseudomonas sp. JS100 (JS100), to explore its degradation efficiency of pentachlorobenzene (PeCB) and the potential degradation mechanism under aerobic conditions. [Methods] The NZVI and JS100 reaction system was constructed to degrade PeCB. The concentrations of PeCB and its intermediates in the reaction system were determined periodically by GC-MS. Moreover, the morphology and growth of JS100 were observed. [Results] The NZVI-JS100 reaction system could degrade about 55.4% of PeCB at 36 h. Compared with the single system of NZVI or JS100, it showed better degradation efficiency The reaction process accorded with pseudo-first-order reaction kinetics, and the rate constant was 0.020 48 h–1. Based on the results from GC-MS of the reaction intermediates, the mechanism of PeCB degradation could be rationally hypothesized. At aerobic conditions, hydroxyl radicals were generated from NZVI, and then attacked PeCB, leading to the production of lower chlorinated benzenes. While JS100 used them as nutrient for growth successively. At the same time, it provided multiple attachment sites and reduced the aggregation of NZVI, and thus, improving the efficiency of the NZVI-JS100 system. [Conclusion] The NZVI-JS100 system synthesized in this study exhibited a high degradation efficiency for PeCB. Moreover, NZVI-JS100 system provided an important reference for the remediation of organic pollutants such as higher chlorinated benzenes in complex environments.