This study determines the inhibitory effect of Stevia rebaudiana leaf extracts and its purified bioactive compound ‘stevioside’ against food-related pathogens. The S. rebaudiana solvent extracts (1000 μg/mL) displayed antibacterial activity to Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, B. subtilis, Alcaligenes denitrificans and Salmonella typhimurium. Of the six solvents, ethanol and acetone extracts displayed the highest zone of inhibition. The bioactive compound from S. rebaudiana was purified by solvent extraction, thin-layer chromatography followed by structural characterization by spectroscopy evidence. Purified stevioside prevented the growth of tested bacterial species, i.e. B. subtilis, K. pneumoniae and S. typhimurium. Significant zone of inhibition (12 mm) was observed against B. cereus which proposes potential application of stevioside in foods to increase their shelf life.