Milk is a universal food that is widely consumed around the world irrespective of age. It has a complex matrix consisting of proteins, carbohydrates, minerals, vitamins, and enzymes that have successfully retained the interest of researchers as yet. The aim of the study was to evaluate the effect of low-temperature long-time (LTLT), ultraviolet light (UV), and ultrasound (US) treatments on physicochemical properties, microbial count, and fatty acid profiling of three types of bovine milk – Country (C), Jersey (J), and Holstein Friesian (HF). The study was conducted in southern India, where these three types of milk are commonly consumed. It was found that no significant (p ˂ 0.05) differences in the proximate composition of milk samples subjected to different treatments compared to untreated milk. However, LTLT treatment resulted in a reduction in ascorbic acid content and significant changes in antioxidant activity and lactose content. LTLT treatment was also able to kill the pathogenic bacteria E. coli, whereas UV and US treatment only showed a notable reduction in the E. coli count. Fatty acid profiling of the treated milk samples was done using GC/MS, and Fourier Transform Infrared Spectroscopy (FTIR) was performed to study the types of chemical bonds present. The results showed that non-thermal treatments (UV and US) were able to sustain the chemical characteristics of milk and had potential for implementation in the dairy industry. Overall, the study highlights the potential benefits of non-thermal treatments such as UV and US in maintaining the chemical properties of milk and reducing pathogenic bacteria count. The findings could be useful in the dairy industry to ensure the safety and quality of milk.