Tween 80 is a well-known non-ionic emulsifier which is used in pharmaceutical and food industries. Due to its widespread applications it is needed to understand how it affects bacteria. The current study aimed to investigate the effect of Tween 80 on the fatty acid content of bacteria. Three bacterial isolates were used in this study: two isolates of Serratia marcescens and one isolate of Acinetobacter baumannii. The analysis of fatty acid components of bacterial lipids was performed, followed by the assessment of the effect of Tween 80 on fatty acid content by adding it to the culture medium in three concentrations: 0. 1%, 0. 5%, and 1. 0 %. The results indicated that the Tween 80 has the ability to change the fatty acid content present in these bacteria by the appearance and disappearance of three fatty acids, including Elaidic acid 18. 1 trance 9, Oleic acid 18. 1 cis 9, and erucic acid 22. 1, which belong to Mono-Unsaturated Fatty Acids (MUFA), in the presence of different concentrations of Tween 80. The results also demonstrated that the p-value was significant in two situations, the first belonged to S. marcescens at 0. 5 % concentration for all groups of FAs (Saturated fatty acids, Monounsaturated fatty acids, and polyunsaturated fatty acids), and the second was for S. marcescens just in MUFA in all concentrations of Tween 80.