Fatty Acid Methyl Esters (FAMEs) are considered as a valuable and important biologically active compounds based on their many health benefits in addition with radical scavenging properties. FAMEs were obtained from an endophytic fungus of Eichhornia crassipes (a common water hyacinth). The fatty acid composition was determined in the endophytic fungi strains of two genus which includes Aspergillus terrus, and Curvularia species, using direct trans-esterification method. The FAMEs prepared by direct trans-esterification method was analyzed using Gas Chromatography - Mass Spectrometer Detector). Direct trans-esterification of fatty acid extracts from endophytic fungi mycelial mat and GC-MS analysis has made the identification of fatty acid composition in single step procedure. Totally 49 fatty acids in Curvularia species, and 33 fatty acids in Aspergillus terrus, were determined from GC-MS spectrum observation. The present study was focused on FAME preparation and identification of fatty acid composition using GC-MS analysis and further purification of each fatty acids with their biological activity evaluation is required.