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Abstract Background Currently, cheese fat is a major component of human diet due to change in eating habits. It contains a number of health destroying as well as health promoting fatty acids. Bovine milk cheese fatty acid composition is regulating by many factors. These may be breed of animal, animal health condition like mastitis and stage of lactation. It also differs with feed and dietary fat intake and seasons. Many studies demonstrated physicochemical, textural and sensory characteristics of Mozzarella cheese with variation in technological process but no literature found about the fatty acid profile and potential influence of milling pH on the fatty acid composition of buffalo Mozzarella cheese. Methods Buffalo Mozzarella cheeses were manufactured at 5.2, 5.1, 5.0, 4.9 and 4.8 milling pH, vacuum packaged and stored at 4 °C and analyzed for quality characteristics, mineral composition and fatty acid profile on days 1, 45, and 90. Results were analyzed by ANOVA according to complete randomized design. Results This study evaluated the effect of milling pH on chemical composition, mineral and fatty acid profile of buffalo Mozzarella cheese. Experimentally induced milling pH differences persisted and significantly affected chemical composition during first day of manufacturing but have no effect on fatty acid profile of cheese. However, storage effects significantly on chemical composition and fatty acid profile of cheese. Decreasing milling pH from 5.2–4.9 resulted in decrease in moisture content of cheese. As a result of changes in milling pH, all the cheeses experienced a significant loss in protein content. In contrast to protein content, fat content of cheese increases with decreasing milling pH. Ash contents of cheese decreased with decreasing milling pH. The level of calcium decreases from 77.82 mg/g to 69.1 mg/g with decreasing milling pH while there is no clear trend observed for potassium and sodium during change in milling pH. Saturated fatty acids presented higher concentrations reaching values of about 71.38 g/100 g throughout storage while monounsaturated fatty acids decreases with storage from 26.72 to 22.06 g/100 g. On the other hand, total polyunsaturated fatty acids exhibited lower concentrations than total monounsaturated fatty acids reaching values of 3.2 g/100 g and its value also decreased with ripening and reached to 1.6 g/100 g. Concentration of C18:1 t10–11 was observed 1.89% in freshly prepared cheese. Milling pH did not influence C18:1 t10–11 concentration but storage days significantly (p