Lindane (γ-hexachlorocyclohexane, γ-HCH) is a recalcitrant and toxic organochlorine insecticide. Due to its non-selective production process and widespread use, HCH isomers and their degradation products have been detected frequently in soils and groundwater. An innovative technology using microbial produced Pd(0) nanoparticles, i.e. bio-Pd, was developed to treat groundwater containing a mixture of HCHs and chlorobenzenes. In a first step, the groundwater was de-ironized and most of the chlorobenzenes were removed in a biological trickling filter. The μg L−1 levels of HCHs and chlorobenzenes were removed in a second step by the bio-Pd-based technology. Therefore, a 200-L pilot scale reactor was developed with 100 mg L−1 bio-Pd encapsulated in alginate beads. Hydrogen gas was bubbled at the bottom of the reactor and served to charge the bio-Pd catalyst. The reactor influent contained 5.2 μg L−1 HCHs and 51.1 μg L−1 chlorobenzenes. During a test period of 10 days, 29% of the HCH isomers and 63% of the chlorobenzenes were removed applying a nominal hydraulic residence time of 4 h. These removal percentages could be increased to 75 and 68% by doubling the nominal hydraulic residence time to 8 h. This study demonstrated that biologically produced nanoparticles of Pd can be applied for the large-scale remediation of groundwater contaminated with HCHs.