In this study, performance evaluation for the gas-phase o-xylene removal using a xylene-acclimated biotrickling filter (BTF) was conducted. Substrate interactions during aerobic biodegradation of three poorly soluble compounds, both individually and in paired mixtures (namely, o-xylene and ethyl acetate, o-xylene and dichloromethane, which are common solvents used by pharmaceutical industry), were also investigated. Experimental results indicate that a maximum elimination capacity of 99.3 g x m(-3) x h(-1) (70% removal) was obtained at an o-xylene loading rate of 143.0 g x m(-3) x h(-1), while the top packing layer (one-third height of the three packing layers) only contributed about 13% to the total elimination capacity. Kinetic constants for o-xylene biodegradation and the pattern of o-xylene removal performance along the height of the BTF were obtained through the modified Michaelis-Menten kinetics and convection-diffusion reaction model, respectively. A reduction of removal efficiency in o-xylene (83.2-74.5% removal at a loading rate of 40.3 g x m(-3) x h(-1) for the total volatile organic compound (VOC) loading rate of 79 g x m(-3) x h(-1)) in the presence of ethyl acetate (100% removal) was observed, while enhanced o-xylene removal efficiency (71.6-78.6% removal at a loading rate of 45.1 g x m(-3) x h(-1) for the total VOC loading rate of 90 g x m(-3) x h(-1)) was achieved in the presence of dichloromethane (35.6% removal). This work shows that a BTF with xylene-acclimated microbial consortia has the ability to remove several poorly soluble compounds, which would advance the knowledge on the treatment of pharmaceutical VOC emissions.