Simple Summary: Mastitis is recognized as the most prevalent dairy cow disease in the world, which directly leads to inflammatory damage of dairy cow mammary tissue, a decrease in mammary gland cell secretion function, and an increase in somatic cell count, thus affecting milk production and quality, and resulting in huge economic losses in the dairy cow breeding industry. Previous researchers have explored the pathogenesis and metabolism of dairy cows with mastitis. However, information on the relationship between microorganisms and lipids in mastitis is limited. This study attempted to explore the relationship between the microorganisms and lipids of dairy cows with mastitis using a 16S rDNA sequencing technology and lipidomics technique. Based on the obtained results, we found that microorganisms caused abnormal lipid metabolism through positive or negative regulation of lipid metabolites, and ultimately decreased milk quality. Dairy mastitis is an inflammatory reaction caused by mechanical injury and stress within the mammary gland, during which microbial changes and abnormal lipid metabolism occur. However, the underlying mechanism is still unclear. The present study used a combination of 16S rDNA sequencing technology and lipidomics techniques to reveal the effects of mastitis on lactic microbiota and metabolites in the milk of dairy cows. Twenty multiparous Holstein dairy cows (2–3 parities) with an average body weight of 580 ± 30 kg were selected for this study. The dairy cows were allocated to control group (<5 × 104 cells /mL)) and mastitis group (>5 × 106 cells /mL) based on the somatic cell count. The results showed that mastitis caused a decrease trend in milk production (p = 0.058). The results of the 16 s sequencing indicated a significant decrease (p < 0.05) in the number of Proteobacteria, Tenericutes colonized in mastitis milk, and the number of Firmicutes, Bacteroidetes and Actinobacteria communities increased significantly (p < 0.05). The lipidomics results revealed that the changes in lipid content in mastitis milk were correlated with arachidonic acid metabolism, α -linolenic acid metabolism and glycerol phospholipid metabolism. The results showed that mastitis may cause abnormal lipid metabolism in milk by regulating the diversity of milk microflora, and ultimately affect the milk quality. [ABSTRACT FROM AUTHOR]