Diffusion, sorption-desorption, and biodegradation influence chlorinated solvent storage in, and release (mass flux) from, low-permeability media. Although bioenhanced dissolution of non-aqueous phase liquids has been well-documented, less attention has been directed towards biologically-mediated enhanced diffusion from low-permeability media. This latter process was investigated using a heterogeneous aquifer cell, packed with 20–30 mesh Ottawa sand and lenses of varying permeability (1.0 × 10⁻¹²-1.2 × 10⁻¹¹ m²) and organic carbon (OC) content (<0.1–2%), underlain by trichloroethene (TCE)-saturated clay. Initial contaminant loading was attained by flushing with 0.5 mM TCE. Total chlorinated ethene removal by hydraulic flushing was then compared for abiotic and bioaugmented systems (KB-1® SIREM; Guelph, ON). A numerical model incorporating coupled diffusion and (de)sorption, facilitated quantification of bio-enhanced TCE release from low-permeability lenses, which ranged from 6 to 53%. Although Dehalococcoides mccartyi (Dhc) 16S rRNA genes were uniformly distributed throughout the porous media, strain-specific distribution, as indicated by the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA, was influenced by physical and chemical heterogeneity. Cells harboring bvcA comprised 44% of the total RDase genes in the lower clay layer and media surrounding high OC lenses, but only 2% of RDase genes at other locations. Conversely, cells harboring the vcrA gene comprised 50% of RDase genes in low-permeability media compared with 85% at other locations. These results demonstrate the influence of microbial processes on back diffusion, which was most evident in regions with pronounced contrasts in permeability and OC content. Bioenhanced mass transfer and changes in the relative abundance of Dhc strains likely impact bioremediation performance in heterogeneous systems.