Malachite green (MG), a suggestive chemical for tumor development and carcinogenicity, is widely used as an illicit food coloring agent, posing risks to consumers and handlers. This study aimed to assess the toxic effects of MG in both plant and animal models. Different doses of MG (375, 750, and 1500 mg/L) were applied for 24 h to evaluate root growth inhibition, mitotic index (MI), and chromosomal aberrations in Allium cepa L. roots for genotoxicity analysis. In animal studies, forty Swiss albino mice were divided into four groups: control and three treatment groups, which were orally administered MG at 375 (low), 750 (medium), and 1500 (high) mg/kg body weight for 13 weeks. Hematological, biochemical, histopathological, and molecular analyses were performed on liver, kidney, and intestinal tissues post-treatment. MG significantly reduced root length and MI in A. cepa roots dose-dependently causing chromosomal abnormalities. MG treatment significantly lowered the body weights of mice and increased platelet, monocyte, and white blood cell counts, while reducing hemoglobin, hematocrit, and red blood cell counts. Serum analysis showed elevated ALT, ALP, AST, bilirubin, creatinine, and urea, indicating hepatotoxicity and nephrotoxicity. Histopathological examination revealed vacuolation, congestion, and inflammatory infiltration in the liver, glomerular shrinkage, tubular degeneration, and interstitial edema in the kidney, and epithelial sloughing, submucosal necrosis, and inflammatory infiltration in the colon. RT-qPCR analysis demonstrated increased Bcl-2, Beclin-1, and NF-κB mRNA expression with decreased Bax mRNA. These findings suggest MG is a potent genotoxic and carcinogenic agent even at lower doses threatening human health.