The potential for bioaccumulation and associated genotoxicity of nonextractable residues (NERs) of polycyclic aromatic hydrocarbon (PAHs) in long-term contaminated soils have not been investigated. Here we report research in which earthworms, Eisenia fetida, were exposed to a soil containing readily available benzo[a]pyrene (B[a]P) and highly sequestered B[a]P NERs aged in soil for 350 days. B[a]P bioaccumulation was assessed and DNA damage (as DNA single strand breaks) in earthworm coelomocytes were evaluated by comet assay. The concentrations of B[a]P in earthworm tissues were generally low, particularly when the soil contained highly sequestered B[a]P NERs, with biota-soil accumulation factors ranging from 0.6 to 0.8 kg/kg. The measurements related to genotoxicity, that is percentage (%) of DNA in the tails and olive tail moments, were significantly greater (p < 0.05) in the spiked soil containing readily available B[a]P than in soil that did not have added B[a]P. For example, for the soil initially spiked at 10 mg/kg, the percentage of DNA in the tails (29.2%) of coelomocytes after exposure of earthworms to B[a]P-contaminated soils and olive tail moments (17.6) were significantly greater (p < 0.05) than those of unspiked soils (19.6% and 7.0, for percentage of DNA in tail and olive tail moment, respectively). There were no significant (p > 0.05) differences in effects over the range of B[a]P concentrations (10 and 50 mg/kg soil) investigated. In contrast, DNA damage after exposure of earthworms to B[a]P NERs in soil did not differ from background DNA damage in the unspiked soil. These findings are useful in risk assessments as they can be applied to minimise uncertainties associated with the ecological health risks from exposure to highly sequestered PAH residues in long-term contaminated soils.