The present study assessed the toxicological, biochemical, and mechanism of action of Colocasia esculenta leaf extract (CELE) on Wistar albino rat and on cholinergic, antioxidant, and antiinflammatory enzymes in Sitophilus zeamais. This was with a view to assessing the potential benefits and safety profile of CELE as a natural alternative for insect control. The bioactivity of the fraction was evaluated using insecticidal and repellent activities against colonies of Sitophilus zeamais to obtain a VLC-chromatographed fraction which was spectroscopically characterized and investigated for enzyme inhibition. The results revealed the ethyl acetate fraction (EAF) as the most potent one with LC50 6.198 μg/ml and 6.6 ± 0.5 repellency. The EAF had an LD50 > 5000 mg/kg but repeated dose >800 mg/kgbw po administration caused significant (p < 0.05) increase in liver and kidney function biomarkers accompanied with elevated atherogenic and coronary indices. Also, renal and hepatomorphological lesions increased in a dose–dependent manner. The High-Performance Liquid Chromatography analysis profiled 7 unknown compounds while the GC-qMS revealed 103 compounds in the CC6 fraction allowing for their identification, quantification, and providing insights into the biological activities and its potentials application. The CC6 fraction inhibited glutathione S-transferase (IC50 = 2265.260.60 mg/ml), superoxide dismutase (IC50 = 1485.300.78 mg/ml), catalase (IC50 = 574.471.57 mg/ml), acetyl cholinesterase (IC50 = 838.280.51 mg/ml), butyryl cholinesterase (IC50 = 1641.76 ± 1.14 mg/ml) and upregulated cyclooxygenase-2 (IC50 = 37.89 ± 0.15 mg/ml). Based on the result of the study, it could be inferred that the unidentified compounds present in the EAF exhibit strong insecticidal properties. The study concluded that the acute toxicity of the potent fraction showed no abnormal clinical toxic symptoms while a repeated dose of the extract in sub-acute studies showed a toxic effect that is dose-dependent. The mechanism of action of the purified fraction could be said to be by inhibition of cholinergic and antioxidant enzymes. However, the potent fraction also upregulated the activity of anti-inflammatory enzymes. Hence, regulated amount of CELE at a repeated dose <800 mg/kgbw could be considered for use as an anti-pest agent in Integrated Pest Management of Sitophilus zeamais.