AbstractAflatoxin M₁ (AFM₁) is a significant contaminant of food, particularly dairy products and can resist various industrial processes. Several probiotic strains like Lactobacillus plantarum are known to reduce aflatoxin availability in synthetic media and some food products. The current work investigated the possible chitosan coating prophylactic efficacy of Lactobacillus plantarum RM1 nanoemulsion (CS-RM1) against AFM₁-induced hepatorenal toxicity in rats. Twenty-eight male Wistar rats were divided into four groups (n = 7) as follows: group 1 received normal saline, group 2 received CS-RM1 (1mL contains 6.7 × 10¹⁰ CFU), group 3 received AFM₁ (60 µg/kg bwt), and group 4 received both CS-RM1(1 mL contains 6.7 × 10¹⁰ CFU) and AFM₁ (60 µg/kg bwt). All receiving materials were given to rats daily via oral gavage for 28 days. AFM₁ caused a significant elevation in serum levels of ALT, AST, ALP, uric acid, urea, and creatinine with marked alterations in protein and lipid profiles. Additionally, AFM₁ caused marked pathological changes in the liver and kidneys, such as cellular necrosis, vascular congestion, and interstitial inflammation. AFM₁ also increased the MDA levels and decreased several enzymatic and non-enzymatic antioxidants. Liver and kidney sections of the AFM₁ group displayed strong caspase-3, TNF-α, and iNOS immunopositivity. Co-treatment of CS-RM1 with AFM₁ significantly lowered the investigated toxicological parameter changes and markedly improved the microscopic appearance of liver and kidneys. In conclusion, AFM₁ induces hepatorenal oxidative stress damage via ROS overgeneration, which induces mitochondrial caspase-3-dependent apoptosis and inflammation. Furthermore, CS-RM1 can reduce AFM₁ toxicity in both the liver and kidneys. The study recommends adding CS-RM1 to milk and milk products for AFM₁-elimination.