This research aimed to study the effect of various growth conditions (temperature, culture age and culture medium) on the fatty acids composition of cytoplasm membrane of 37 isolates of Staphylococcus aureus using Gas Chromatography-Mass Spectrometry (GC-MS). Results revealed that the total number of fatty acids of Staph. aureus was 33. Eight of them were principle and included: anti-isotetradecanoic acid (aC15:0), octadecanoic acid (C18:0), eicosanoic acid (C20:0), isotetradecanoic acid (Ci14:0), isotridecanoic acid (Ci15:0), hexadecanoic acid (C16:0), antisohexa- decanoic acid (Ca17:0) and octadecenoic acid (C18:1) with a percentage of 33.96, 18.37, 14.94, 7.32, 6.23, 4.14, 3.03 and 2.89%, respectively. The percentage of each of the other fatty acids was less than 1%. Increasing the incubation temperature was associated with reducing the percentages of Ca15:0, Ci15:0, C16:0 and C18:1; and increasing Ca17:0, Ci14:0, C18:0 and C20:0. The increase in incubation period caused a decrease in C16:0, C18:0, C18:1 and C20:0; and an increase in Ca15:0 and Ca17:0. Moreover, the variation in culture media caused differences in the percent of Ca15:0, C18:0 and C20:0; while the other fatty acids showed no changes.