Application of subcritical water (sub-CW) treatment on the yield of oil and fat produced from squid waste entrails was studied experimentally in the temperature range of 443-653 K with a reaction time of 1-40 min. This process showed that the oil and the fat phases contain nine free fatty acids which were produced during hydrolysis of the triglyceride molecules and showed a maximum total yield at 513 K and 40 min (0.44 kg/kg oil). The order of the yields for the nine fatty acids, including omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), was found to be DHA > palmitic > oleic > EPA > others. On the other hand, transesterification of the oil and the fat phases in the presence of trimethylsulfonium hydroxide (TMSH) which converted the free fatty acids to the corresponding fatty acid methyl esters (FAMEs) was studied. The total yield of FAMEs in the oil and the fat phases illustrated a peak at 473 and 493 K, respectively.