Cultivation based and culture independent molecular approaches were used to characterize the composition and structure of bacterial community from a natural warm spring in the Western Ghats, a biodiversity 'hotspot'. Dilution plating was done on three types of media with varying nutrient levels. Relatively nutritionally poor medium supported growth of highest number of bacteria (4.98 x 10(3) ml(-1)) compared to nutritionally rich media. On the basis of different morphological features on the plate, 62 aerobic and heterotrophic bacterial strains were isolated and their 16S rRNA genes were sequenced and analyzed. On the basis of sequence similarity these isolates were found to be distributed in 21 different genera belonging to Proteobacteria (58%) followed by Firmicutes (26%), Actinobacteria (13%) and Bacteroidetes (3%). Amplification of 16S rRNA gene of the community DNA using eubacterial primers, followed by cloning and sequencing revealed that predominant members of the habitat belong to the phylum Cyanobacteria (60%) followed by Proteobacteria (19.5%), Bacteroidetes (6.67%), Actinobacteria (4.4%) and Firmicutes (2.2%) and small ribosomal subunit of a plastid (of Chlorophyta, 2.2%).