Hepatic steatosis/non‐alcoholic fatty liver disease is a major public health delinquent caused by the excess deposition of lipid into lipid droplets (LDs) as well as metabolic dysregulation. Hepatic cells buildup with more fat molecules when a person takes high fat diet that is excessive than the body can handle. At present, millions of people in the world are affected by this problem. So, it is very important to know the effects of factors responsible for the disease. Here, the role of lipid droplet (LD) biogenesis and metabolism was analyzed and intended to investigate if defects in biogenesis/metabolic enzymes are responsible for the accumulation of lipids other than LDs in fatty liver disease in high‐fat‐induced conditions in mice model. To explore it, high‐fat diet (HFD), fast food (FF), and soft drinks (SD) were administered to wild‐type Swiss albino mice for 14 weeks following yogurt supplementation. After experimental period, glucose tolerance, enzyme function, lipid profile, plasma biochemistry, and other analytical tests were analyzed by auto‐analyzer including different oxidative stress markers. Lipids from hepatic tissues were extracted, and purified by Floatation Assay and subsequently analyzed by different biochemical and chromatographic techniques. Histological architecture of hepatocytes was performed using Zeiss microscope. Finally, increased amount of lipids biogenesis/accumulation was found in liver tissues that causes Fatty liver disease. Significantly, HFD, FF, and SD were identified as factors for the increased LD biogenesis and or lipid metabolic disorder. Nevertheless, yogurt supplementation can homeostasis those LD formation and metabolic syndrome as it increases the down regulation of lipid biogenesis as well as lipid metabolic rate. So, yogurt supplementation was considered as a novel agent for decreasing LD biogenesis as well as excessive accumulation of fat in hepatocytes which can be used as therapeutics for the treatment of NAFLD.