Polyunsaturated fatty acids offer a wide variety of health advantages. Fourteen yeast isolates were obtained from different dairy products. Preliminary lipid production screening revealed that five yeast isolates were capable to accumulate lipids within its cells, as detected by Sudan black B staining. Quantitative analysis of lipid content after yeast growth on a nitrogen-limited medium showed significant variations of lipid content among the yeast isolates. Notably, majdoula cheese isolate presented the highest lipid content 27.69%, followed by rayeb milk, labneh, mozzarella cheese, and ras cheese isolate recorded 22.73%, 22.37%, 21.79%, and 8.04%, respectively. Furthermore, Gas chromatography of fatty acids revealed that ras cheese yeast isolate exhibited oleic acid as the abundant unsaturated fatty acid, afterward linoleic acid, linolenic acid, palmitoleic acid, homo-γ-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid sequentially, and it represents about 64.9 % of all fatty acids. Similarly, mozzarella cheese isolate and rayeb milk isolate showed a predominance of oleic acid, linoleic acid, and palmitoleic acid, representing over 62% and 56% of the total fatty acids, respectively. Three yeast isolates with the highest unsaturated fatty acids content were identified as Pichia kudriavzevii PP527343, Yarrowia lipolytica PP527342, and Yarrowia lipolytica PP701998 by 18s rRNA.