Pine wilt disease is caused by a complex of the pine wood nematode (PWN), Bursaphelenchus xylophilus and its associated bacteria. To isolate the aseptic nematode, the sterilizing effect of seven chemicals on PWN and its accompanying bacteria Burkholderia cepacia (strain B619) in a series of concentration and treating time were investigated. B619 could be completely killed by peroxide, sodium hypochlorite, mercuric chloride, malachite green, and gentamycin and couldn’t by lysozyme and streptomycin. PWNs could not be disinfected effectively by any of these chemicals. However, The survival rate of PWN decreased as the treatment time and concentration of these disinfectants were increased. Treatments at room temperature (22 °C to 26 °C) or at low temperature (4 °C) improved the eliminating effect of 0.1% mercuric chloride, 3% peroxide, and 4% sodium hypochlorite. Higher PWN survival was observed at low temperatures compared with that at room temperature. The chemicals that most effectively eliminated the bacteria and decreased the PWN survival rate were 3% peroxide, 0.1% mercuric chloride, and 4% sodium hypochlorite. The results suggested that PWN might have a protective effect on its associated bacteria.