Regular and irrational use of insecticides have adverse effects on the fauna below and above the ground surface. In environmental monitoring studies and toxicity assays, earthworms are an ideal biological model. The survival, growth and reproductive parameters are considered as indicators of effect of insecticide toxicity upon the organism. The present study compared the imidacloprid affects on the reproductive performance and genotoxicity in Eudrilus eugeniae and Metaphire posthuma. The LC₅₀ value was calculated by exposing earthworms to different concentrations of imidacloprid (1.00, 2.50, 4.00, 5.50 and 7.00 mg/kg dry soil). The LC₅₀ value calculated was 3.19 mg/kg and 2.23 mg/kg for earthworm E. eugeniae and M. posthuma, respectively. In artificial test soil, E. eugeniae and M. posthuma were exposed to doses 0.3, 0.6 and 1.0 mg/kg to evaluate reproductive potential along with genotoxicity studies as per OECD guidelines. Mortality due to morphological alterations was 3.33% and 10% for E. eugeniae and M. posthuma respectively in 1.0 mg/kg dose. Cocoon production and hatchling success decreased from lower (0.3 mg/kg) to higher dose (0.6 and 1.0 mg/kg) and was nil in 1.0 mg/kg. Comet assay revealed a high DNA damage in both the earthworm species which increased significantly (p < 0.05) in dose (0.6 and 1.0 mg/kg). These laboratory studies revealed that the effects of imidacloprid are species specific as well as dose and duration-dependent. M. posthuma is more susceptible as compared to E. eugeniae leading to loss of soil fauna.