The discoveries of potential anti-inflammatory agents are still needed to mitigate the inflammation mediated cardiovascular and neurodegenerative disorders. Many anti-inflammatory compounds were discovered from plant sources, but very little research is done based on animal sources. This study discovered a Novel Bufotenin Variant (NBV), which was not reported earlier, from paratoid gland venom of Indian Toad with potential anti-inflammatory activities. NBV was characterized by IR, 1H NMR, 13C NMR, LC-MS, P-XRD, COSY, HSQC and HMBC, and in silico molecular docking demonstrated that, proteins for PDB-2L3Y (IL-6) and 2AZ5 (TNF-α) with a stronger binding affinity. Due to limited information on investigation of protective activities with different inducers such as λ - carrageenan induced paw edema and air pouch mouse models, Bisphenol-A is used for kidney and hepato toxicity and colitis induced by Dextran Sodium Sulphate (DSS) for in vivo studies and NBV treated groups significantly inhibited TNF-α and IL-6, and further confirmed by histological and immunohistochemical studies. Paw edema, liver, kidney and colon tissue, serum levels of IL-6 and TNF-α significantly decreased with high dose of NBV (20mg/kg).This study expedited that NBV as a potent protective agent with the mechanism of action by inhibition of both TNF-α and IL-6. Further NBV will become a new chemical entity with protective properties can attract the researchers to discover better anti-inflammatory drugs and it may help in the new drug discovery for the treatment of many inflammations mediated diseases. Further systematic studies are needed to get the potential therapeutic benefits of NBV.