This study aims to improve encapsulation and stability of eicosapentaenoic acid and docosahexaenoic acid by optimizing high-pressure homogenization conditions of monolayered (lecithin + maltodextrine) and multilayered (lecithin + chitosan-maltodextrine) fish oil emulsions. First, a positive influence of high-pressure homogenization on quality characteristics of emulsion and microcapsules was observed. Trials were conducted to get optimum combination conditions of pressure and number of passes of homogenization of both types of emulsions for improving microencapsulation yield and efficiency and lipid oxidation of the corresponded microcapsules. These studied variables showed a notable effect on monolayered microcapsules, being less influencing on multilayered ones. Finally, 1,200 Ba—3 passes and 1,100 Ba—2 passes were selected as optimum combination for monolayered and multilayered emulsions, respectively. This led to microcapsules with improved quality characteristics in terms of microencapsulation yield, microencapsulation efficiency, and oxidation stability, especially in the case of monolayered microcapsules. Practical applications: The results of this study suppose an advance in the investigations about microencapsulation of bioactive compounds, being specifically useful to develop high stable vehicles of omega-3 fatty acids to enrich food products. To the best of our knowledge, this kind of homogenization procedure has been used to elaborate fish oil microcapsules from monolayered (lecithin + maltodextrine) and multilayered (lecithin + chitosan-maltodextrine) emulsions. The knowledge of the optimal homogenization conditions is important to achieve high quality microcapsules in terms of oxidative stability and efficiency of encapsulation.