The present study was focused on distribution of microbial and diazotrophic Azotobacter and Azospirillum population in rhizosphere soil samples. Total microbial population viz, bacteria, fungi, actiomycetes and diazotrophic Azotobacter and Azospirillum were enumerated from five different rhizosphere soil samples and compared the distribution of diazotrophic Azotobacter and Azospirillum with other three microbial groups. The result of enumeration of total microbial population indicates the higher presence of bacterial population followed by Azotobacter and Azospirillum population in various rhizosphere soil samples. Based on the predominant growth, five strains of Azotobacter and five strains of Azospirillum (one isolate from each sample) were isolated and identified through morphological and biochemical characteristics. In order to asses the nitrogen fixing potential of Azotobacter and Azospirillum isolates, Acetylene Reductase Assay(ARA) was carried out using Gas Chromatography(GC).The results of GC-ARA reveals that Azotobacter strain-5 and Azospirillum strain-1 exhibit better nitrogen fixing potentials than other strains. All the ten diazotrophic bacterial strains were authenticated through SEM imaging and 16S rRNA phylogenetic tree analysis. Based on Neighbor joining algorithm using NCBI BLAST tool, all the ten diazotrophic bacterial strains were authenticated viz., Azotobacter nigricans, A. beijerinckii, A. chroococcum, A. vinelandii, A. chroococcum, Azosprillum fermentarium, A. humicireducens, A. thiophilum, A. melinis, A. canadens
