In this study, seven strains of cells were isolated from a trickle-bed air biofilter used for continuously treating paint of volatile organic compounds (VOCs) for six months. The morphology and biochemical study was conducted by streaking isolated mixed culture in solid agar slant media and the cell shapes were identified by using an electron microscope. It was found that this mixed culture was gram-negative for seven different isolates. The isolated strains were grown on substrates including methyl ethyl ketone (MEK), toluene, n-butyl acetate, and o-xylene (MTBX), as the carbon and energy sources. Among the seven isolates, an AKM 02 strain had a high MTBX-degrading activity and was identified as Shewanella putrefaciens by taxonomical analysis, biochemical tests and 16S rDNA gene analysis methods. All isolates grew in a pH range from 3.0-11.0 with an optimum range of 6.0-8.0. In addition, each of the isolates grew in the temperature range of 15-45 degrees C with an Optimum range between 25-30 degrees C. The batch experiments were conducted at four different initial MTBX concentrations ranging from 100-1000 mg L-1. S. putrefaciens was capable of completely degrading n-butyl acetate, MEK and toluene at a concentration lower than 500 mg L-1. When the initial concentration of n-butyl acetate, MEK and toluene were 500 mg L-1, S. putrefaciens could remove the n-butyl acetate, MEK and toluene completely within 70, 98 and 110 h, respectively. However, for o-xylene at concentration of 500 mg L-1, incomplete degradation was observed with only 70 % of the o-xylene degraded after 135 h. Collectively, the results indicate that the S. putrefaciens strain degrades MTBX at a faster rate, and this strain can be used effectively in trickle-bed air biofilters for treating high strength mixtures of paint solvents.