Pentachlorophenol (PCP) has been used in the past as a pesticide, herbicide, antifungal agent, bactericide, and wood preservative. Thus, PCP is among the most ubiquitous chlorinated compounds found in groundwater contamination. A former pesticide manufacturing plant located in southern Taiwan has been identified as a PCP spill site. In this study, groundwater samples collected from the PCP site were analyzed to assess the occurrence of natural PCP biodegradation. Microcosm experiments were conducted to (1) evaluate the feasibility of biodegrading PCP by indigenous microbial consortia under aerobic and cometabolic conditions, and (2) determine the potential of enhancing PCP biodegradation using cane molasses and biological sludge cake as the substitute primary substrates under cometabolic conditions. The inocula used in this microcosm study were aquifer sediments collected from the PCP site and activated sludges collected from the municipal and industrial wastewater treatment plants. Results from this field investigation indicate that the natural biodegradation of PCP is occurring and causing the decrease in PCP concentration. Microcosm results show that the indigenous microorganisms can biodegrade PCP under both aerobic and aerobic cometabolism conditions. A PCP-degrading bacterium was isolated from the collected aquifer sediments and identified as Pseudomonas mendocina NSYSU via some biochemical tests and further conformation of DNA sequencing. In batch cultures, P. mendocina NSYSU used PCP as its sole source of carbon and energy. The isolated bacterium, P. mendocina NSYSU, was capable of completely degrading PCP as indicated by the increase in biomass formation with the decrease in PCP concentrations occurred in the carbon-free medium simultaneously. Results indicate that the in situ or on-site aerobic bioremediation using indigenous microorganisms or inoculated bacteria would be a feasible technology to clean up the studied PCP-contaminated site. Results from this study will be useful in designing a scale-up in situ or on-site PCP bioremediation system (e.g., on-site bioreactor) for field application.