Analysis of fatty acid (FA) is one of the most commonly used tools for investigating microbial populations in ecological studies. Fatty acids can be extracted and esterified to form fatty acid methyl esters (FAME) when analyzed using gas chromatography-mass spectrometry, the resulting profile contains some microbial biomarkers. The aim of the present study to analysis FAME present in two species of earthworms exhibit the same environment. The thin layer chromatography of earthworm Allolobophora caliginosa Savigny showed three major spots corresponding to FAME while that of earthworm Pheretima hawayana Rosa showed two major spots. The GC-MS analysis of Allolobophora caliginosa Savigny extract showed the presence of at least 23 peaks, only two peaks were identified from their Rt and Ms spectrum, hexadecanoic acid methyl ester and octadecenoic acid methyl ester. While the chromatogram of Pheretima hawayana Rosa extract showed at least 20 peaks, five of them were identified; butanedioic acid dimethyl ester, pentadecanoic acid 1 4-methyl ester, 2-hydroxy-hexadecanoic acid methyl ester, 9-octadecenoic acid methyl ester and octadecanoic acid methyl ester. In conclusion, in spite of the two species of earthworms from the same environment, but their content of FAME was different and that may explain why the biological activity of the whole body extract was different.