Objectives: To identify the bacteria, fungi and actinomycetes isolated from soil samples augmented with biochar by fatty acid methyl ester method. Methods and Statistical analysis: The fatty acid profiles of microbial populations were identified by FAME analysis. Analysis of variance (ANOVA) and principal component analysis of microbial populations were carried out using SYSTAT 9. Findings: Three bacteria, two fungi, three actinomycetes isolated from soil samples augmented with biochar, were identified by FAME analysis based on the presence of particular fatty acid biomarkers. The current study showed that the major fatty acid was 15:0 iso followed by 15:0 anteiso for the bacterial isolates Stenotrophomonas maltophila and Bacillus megaterium. The major fatty acids present in Bacillus cereus was 15: iso followed by 17:0 iso. The results showed that the major fatty acids in fungi (Cladosporium carrionii, Phoma spp) were18:2 CIS 9,12/18:0a followed by summed feature 8 and 16:0. The major fatty acids in actinomycetes was 15:0 anteiso followed by 16:0 iso for Streptomyces-rochei-rochei and 17:0 anteiso for Streptomyces-halstedii-scabies. PCA (principal component analysis) resulted in clear separation of microbial communities. So, it can be used for the comparison of microbial communities based on fatty acid profile. Novelty and applications: Previous studies scarce data of the microbial populations in the rhizosphere soil of rubber in Kerala. The current study helps in the evaluation of major microbial population in the rhizosphere soil of rubber in Kerala by FAME analysis.