The present study was carried out to evaluate the influence of biofertilizers on stevioside content (Main active principle) in the leaves of Stevia rebaudiana grown in acidic soil condition. The six month field experiment study was revealed the combined application of three biofertilizers showed that the total yield of fresh biomass has been recorded an increase up to sixth month, being highest in the combined application of biofertilizers over that of their corresponding sole applications. The percent increase of bio-mass yield was recorded highest (22.14%) in the treatment when all the bio-fertilizers were applied together. Further the results envisaged the content of stevioside were recorded significantly higher (20.17 %) with the same treatments (T8) as compared to control by applied HPLC chromatogram. The chromatographic separations were carried out using a C18 column using the mobile phase consisting of methanol and water, and with UV detection at 210 nm. The limits of determination of stevioside were 4 μg/ml for the leaf extracts.