Much attention has been given recently to the biological activity of unsaturated fatty acids. The present paper concerns the antibiotic action of unsaturated quaternary ammonium salts and their preparation method with a view to utilizing fish oil. The preparation process is as follows: fish oil or methyl ester of fish oil → alcohol → bromide → triethylammonium bromide → pyridinium bromide During the above process, the decrease of iodine value is remarkable (ca. 40%), especially in the alcoholization process (ca. 30%) (table 2). Regarding the antibiotic activity of quaternary ammonium bromides against Candida albicans, the activity of triethylammonium bromides is the same as that of corresponding pyridinium bromides. The activity of unsaturated quaternary ammonium bromides is affected by the carbon chain length and the degrees of unsaturation in the long carbon chain, whereas that of saturated compounds is influenced only by the carbon chain length. Any lengthening carbon chain more than C16 weakens the activity and an increase of unsaturation enhances it. But it seems that these influencing factors tend to interfere with each other, because there is not any remarkable difference in the antibiotic activity of the six unsaturated quaternary ammonium bromides prepared from methyl esters of calamary oil (table 3). Triethylammonium bromide prepared from sardine oil inhibits the growth of fungi at 1:310, 000 except Rhizopus japonicus and that of yeasts and bacteria at 1:1600, 000 except Pseudomonas fluorescens, Proteus vulgaris and Escherichia coli (table 4). It is indicated by comparison that the activity of unsaturated quaternary ammonium bromides is superior to that of unsaturated fatty acids or oxidized fatty acids. The facts that the surface of microorganism is, as a rule, negatively charged, and that the unsaturated group of quaternary ammonium bromides in aqueous solution is found in the positively charged portion, may be regarded to have an important role in causing their highly antibiotic activity.