Spiders are among the most diverse groups of arthropods remarkably known for extra oral digestion. The largest effort based on targeted 16S amplicon next generation sequencing was carried out to decipher the inter subfamily comparison of gut bacterial diversity in spiders and their functional relationship. Twelve spider species belonging to three subfamilies, Araneinae (8), Argiopinae (2) and Gasteracanthinae (2) of family Araneidae have been studied. Analysis revealed the presence of 22 phyla, 145 families, and 364 genera of microbes in the gut microbiome, with Proteobacteria as the highest abundant Phylum. Moreover, the phyla Firmicutes, Actinobacteria and Deinococcus_Thermus were also detected. The bacterial phyla Bacteriodetes and Chlamydiae dominated in Cyclosa mulmeinensis and Neoscona bengalensis respectively. At genera level, Acinetobacter, Pseudomonas, Cutibacterium, Staphylococcus, and Bacillus were the most dominant genera in their gut. In addition to this, the genus Prevotella was observed only in one species, Cyclosa mulmeinensis, and endosymbiont genus Wolbachia generally responsible for reproductive alterations was observed in one spider species Eriovixia laglaizei. Our study revealed that the gut bacterial diversity of the spiders collected from wild are quite different from the diet driven spider gut bacterial diversity as published earlier. A functional analysis revealed the involvement of gut microbiota in carbohydrate, lipid, amino acids, fatty acids and energy metabolism.