The aim of this study was to optimize the production of polyunsaturated fatty acids (PUFAs), using the response surface methodology (RSM), to use the produced PUFAs in kareish cheese processing. PlackettBurman design (PBD) was employed to screen media components that affect PUFAs development (glucose, olive cake, yeast extract, tryptone, MgSO₄, KH₂PO₄, NH₄Cl, agitation speed, incubation time, and pH), and results showed that olive cake and yeast extract, with confidence level > 98%, had a positive effect on PUFAs production. The central composite design (CCD) of the response surface methodology was used to optimize the selected parameters levels where maximum PUFAs production (1790 mg/l) was observed near the mid-point (0) values (concentrations) of olive cake (15 g/l), tryptone (7.5 g/L) and KH₂PO₄ (1.25 g/l). Polyunsaturated fatty acids account for 47.83 % of the total fatty acid profile, according to gas chromatography analysis of the collected PUFAs. The produced PUFAs was encapsulated using whey protein concentrate and maltodextrin, freeze dried, grinded and incorporated in Kareish cheese manufacture. The average particle size of a 0.005% suspension of oil microcapsules was 671.4 nm with a poly dispersity index of 0.611 indicating a moderate stability of the emulsion. The negative zeta potential of the microcapsules particles was -37.6 mv, which is identical to the -42 mv value recorded in the literature for oil emulsions stabilized by whey protein, maltodextrin, and K-carrageenan. The addition of 0.5 % PUFAs-containing microcapsules to Kareish cheese increased antioxidative activity to 38.13 % compared to 30.14 % for the control, as well as Texture profile analysis (TPA) parameters including hardness, cohesiveness, gumminess, and chewiness. The elasticity of the Kareish cheese sample increased slightly by the addition of 0.5% microcapsules, but higher concentration tended to change the elasticity to a brittleness of the cheese structure.