A gnotobiotic microcosm experiment was conducted to study the toxic effects of phenanthrene (PHE) on three nematodes—Caenorhabditis elegans, Mesorhabditis sp. and Acrobeloides sp., also the possibility of PHE depletion by the nematodes were examined. The results showed that the survival rates of these three nematodes decreased gradually with PHE concentration increased. Without PHE exposure, Caenorhabditis elegans could reproduce within 48 hours and 5 mg/L PHE could inhibit the reproduction of Caenorhabditis elegans. While no reproductive patterns was observed with the Mesorhabditis sp. and Acrobeloides sp. due to their longer generation time. After comparing the relative mortality at 24 h exposure, the tolerance order of those nematode was Mesorhabditis sp. ≥ Caenorhabditis elegans ≥ Acrobeloides sp., and the tolerance of Caenorhabditis elegans reduced gradually with the increased PHE concentration. After 48h exposure, Mesorhabditis sp. still had a higher tolerance than Acrobeloides sp., while after 72h exposure, both of them showed similar tolerance to PHE. All nematodes lost the head swing ability after PHE exposure, and the body length of Caenorhabditis elegans and Acrobeloides sp. reduced gradually with the increase of PHE concentration. All nematodes could help to facilitate the depletion of PHE, and no significant difference was found among three nematodes. In conclusion, PHE could restrain the survival, growth and activity of these nematodes, and inhibit the reproduction of the Caenorhabditis elegans. The survival rates of the nematodes were significantly influenced by nematode species, exposure time, PHE concentrations and their interactions. Mesorhabditis sp. showed the highest resistance to PHE pollution. All the three nematodes could contribute to the depletion of phenanthrene in solution.