This study is aimed at assessing culturable diazotrophic bacterial diversity in the rhizosphere of Prosopis juliflora and Parthenium hysterophorus, which grow profusely in nutritionally-poor soils and environmentally-stress conditions so as to identify some novel strains for bioinoculant technology. Diazotrophic isolates from Prosopis and Parthenium rhizosphere were characterized for nitrogenase activity by Acetylene Reduction Assay (ARA) and 16S rRNA gene sequencing. Further, the culture-independent quantitative PCR (qPCR) was performed to compare the abundance of diazotrophs in rhizosphere with bulk soils. The proportion of diazotrophs in total heterotrophs was higher in rhizosphere than bulk soils and 32 putative diazotrophs from rhizosphere of two plants were identified by nifH gene amplification. The ARA activity of the isolates ranged from 40 to 95 nmol ethylene h(-1) mg protein(-1). The 16S rRNA gene analysis identified the isolates to be members of alpha, beta and gamma Proteobacteria and firmicutes. The qPCR assay also confirmed that abundance of nif gene in rhizosphere of these two plants was 10-fold higher than bulk soil.