Effect of nisin on biochemical and microbial quality and shelf life of vacuum packaged rainbow trout (Oncorhynchus mykiss) during 16 days storage at 4 °C was investigated. According to the obtained results, nisin treated fish showed lower (p < 0.05) and acceptable biochemical (Peroxide value, thiobarbituric acid-index, pH, and total volatile base nitrogen) and bacteriological (total viable counts, psychrotrophic viable counts, and lactic acid bacteria) attributes up to 16 days storage at 4 °C compared with those treated without nisin. Furthermore, FAs composition analysis indicated that presence of the nisin preserved nutritional quality of fish lipid, so that nisin treated samples contained higher percentage (p > 0.05) of essential FAs such as eicosapentaenoic acid and docosahexaenoic acid. This study concluded that treatment of the vacuum packaged rainbow trout with nisin resulted in improvement of quality and extension of shelf life of the fish from 12 to 16 days at 4 °C.