Neonicotinoids are potent agonists of nicotinic acetylcholine receptors that exert insecticidal effects by causing abnormal excitation of the nervous system. Neonicotinoids and their metabolites effect in mammals, including humans, have become a concern. In the present study, we evaluated the effects of chronic exposure of two neonicotinoids, acetamiprid (ACE) and clothianidin (CTD), on Caenorhabditis elegans. We used 1, 10, 100, and 1000 µM solutions of nicotine, ACE, and CTD dissolved in 1% dimethyl sulfoxide (DMSO). Bioassays and motility tests, which are neurotoxicity assessments, were performed on the L1‒L2 larvae of wild-type C. elegans. To evaluate the effect of exposure over multiple generations and the correlation between concentrations and generations, the same study was conducted on the second and third generations of the exposed group. The bioassay results showed concentration-dependent adverse effects: body length, maturity rate, and lifetime number of pups decreased for both ACE and CTD for the first generation. In a multi-generation study, the effect intensified with the progression of generations, and the toxicity of both ACE and CTD was cumulative. This effect was more pronounced in breeding studies. The motility test results showed concentration-dependent adverse effects, such as a decrease in the number of behaviors for both ACE and CTD in both tests for the first generation. In a multi-generation study, the effect intensified with the progression of generations, and this effect was more pronounced with ACE exposure. Thus, the chronic exposure to ACE and CTD may cause cross-generational adverse effects, especially on C. elegans reproduction and motion.