Three bacterial strains, Arthrobacter sp. NB1, Serratia sp. NB2 and Stenotrophomonas sp. NB3, were isolated from contaminated sludge by using nitrobenzene as a sole source of carbon and nitrogen. It was observed that all three strains could degrade nitrobenzene at 400 mg/L initial concentration and mixed-cultivation of these strains could enhance the degradation of nitrobenzene compared with mono-cultivation. Mixture design was used for adjusting the proportions of each strain and the optimal ratio of inoculation size was NB1:NB2:NB3 = 4:4:5, where the nitrobenzene degradation percentage was two times higher than for by the single strain. The results of Plackett-Burman design indicated that Mg(2+), Ca(2+), Fe(2+), Zn(2+) and Mn(2+) had a positive effect on the degradation of nitrobenzene, while Cu(2+) and Co(2+) had a negative effect on it.