Biotransformation, a dynamic process conducted by microorganisms, holds promise in modifying natural compounds for enhanced therapeutic potential. In this study, we leverage bacterial systems to catalyze the transformation of hesperidin, obtained from Citrus sinensis, aiming for structural modifications that could optimize its bioactivity and computational targeting against Helicobacter pylori. Multiple bacterial species were employed to perform biotransformation reactions. HPLC and FTIR analyses were used to determine structural modifications and bio-transformed products. The reaction in which hesperidin metabolite was formed was catalyzed by Bacillus spp. The transformed products, along with the original compound, underwent rigorous bioactivity testing to evaluate their potential in combating oxidative stress, inflammation, and diabetes. Employing well-established in vitro methods, we assessed the bio-transformed derivatives for antioxidant efficacy, revealing an impressive 94% inhibition of free radicals compared to hesperidin. In terms of anti-inflammatory activity, the results showcased a substantial 92% inhibition, while the assessment of antidiabetic activity demonstrated a notable 85% inhibition. The hesperidin metabolites were more active than hesperidin in biological activity evaluations. So, the bio-transformed compound derived from hesperidin, along with pure compound, was used to design a computational drug targeting the bacterium H. pylori. Among these two compounds, the bio-transformed product of hesperidin with an alkyl amine exhibited the highest docking energy of - 180.26 kJ/mol, as compared to pure compound. SwissADME provided valuable insights into the compound's drug-likeness like 0.55 bioavailability and 8.41 synthetic accessibility. ProTox-II evaluated different toxicity endpoints with a 0.96 probability of being inactive in cytotoxicity. These findings support the potential of the bio-transformed compound as a promising candidate for further investigation and development as a drug against H. pylori.