Background <p>Insecticides hold crucial roles in both agriculture and healthcare sectors. However, only a fraction of them is utilized for their intended purpose of eradicating target species, the remainder often disperse into the environment, contributing to pollution. Chlorpyrifos (CP), an organophosphate (OP) insecticide, represents one of the most prevalent chemicals used in agricultural and residential settings, known for its neurotoxic properties. This study aimed to assess the detrimental effect of CP on non-targets using <i>Drosophila</i>, a well-known model in toxicology. The present laboratory-based study was performed with <i>D. melanogaster</i>, to observe the effects of CP (oral intake) using various biological parameters such as fecundity, developmental duration, phenotype deformities, gut and Malpighian tissue damage, lifespan, neurobehavior (larval and adult locomotor activity) and larval brain histology. Flies were initially exposed to CP (within a range of 0.5 to 5&#xa0;ppm) for 24 and 48&#xa0;h to determine LC₅₀ through probit analysis, based of which two sublethal concentrations were chosen for setting experiments.</p> Results <p>Significantly lower fecundity (50–75%), delayed development, reduced locomotor activities (~ 28–70%) and decreased lifespan (51–66%) were observed in CP exposed flies. Morphological deformities, damaged gut tissues and Malpighian tubules and mild neural tissue degeneration were also observed in treated <i>Drosophila</i>. However, the adverse effect was less after 10 generations of exposure due to the development of resistance.</p> Conclusion <p>Thus, the study results warn us to rethink the possible consequences of insecticide exposure in the long-term associated with human health as well as the insects of agricultural and medical importance.</p>