There is a greater demand for natural additives when compared with artificial additives. One of the important flavouring agents is vanillin, which is expensive and the demand is high for natural vanillin. Microbial bioconversion, a suitable alternative, was explored in the present work to produce vanillin from isoeugenol using various bacterial strains from aromatic plant roots. Among the eighteen strains, nine isolates showed maximum tolerance to isoeugenol and vanillin analysed by minimum inhibitory concentration and thin layer chromatography. The strains Bacillus spp. C1 and Pseudomonas spp. OSC1 were found to produce maximum yield of vanillin 1.52g/l and 2.43g/l respectively, and resulting in molar yield of 15.2% and 24.3%.Gram negative rod strain, OSC1 showed capability of producing high amount of vanillin in presence of isoeugenol. The strain OSC1 was optimized and was able to produce higher amount of vanillin; 2g/l of vanillin was produced when the bacterial strain OSC1 was subjected to 10g/l of isoeugenol at 96 hours by bioconversion. Vanillin formation was analysed by spectrophotometer and evaluated accurately by gas chromatography. On the basis of 16S rRNA sequence, the strain OSC1 was identified as Pseudomonas spp.