Sources of contamination in a subsurface environment are petrol, diesel fuel, gasoline at oil refineries, underground storage tanks, transmission pipelines, and different industries. The permeable reactive barrier (PRB) is a promising technology to remediate groundwater in-situ. In this study, synthetic groundwater samples containing toluene are treated in three reactor columns by biological processes. PRB-1 consisted of sand and gravel as reactor media, microbial inoculum (bioaugmentation—BA), and nutrients (biostimulation—BS); PRB-2 consisted of sand and gravel as reactor media, microbial inoculum, nutrients, and 12 layers of nonwoven geotextile fabrics; and PRB-3 consisted of only sand and gravel as reactor media (natural attenuation—NA). This study was conducted to assess the impact of geotextile fabric filter, bioaugmentation, and biostimulation on toluene degradation efficiency. After 167 days of treatment, toluene biodegradation efficiencies varied between 88.2% and 93.8% for PRB 1, between 98.0% and 99.3% for PRB 2, and between 14.2% and 68.6% for PRB 3. The effluent toluene concentrations for PRB-2 were less than the guideline value (0.7 mg/L) of the World Health Organization. Reaction rate data were fitted with a first-order kinetic reaction rate model. This study showed that the toluene removal efficiency in the geotextile layered PRB combined with BA and BS process was significantly higher compared to the other processes tested. This lab-scale study introduced a new PRB configuration suitable for the remediation of sites contaminated with toluene.