Plants are the major source of natural flavour ingredients reported for their wide applications in food and pharmaceuticals, oral care and wellness products, etc. We have investigated the water‐soluble fractions (WSF) of basil tetraploid (O. basilicum L.) for their toxicity and biological potential against Salmonella Typhimurium, a pathogen causing around one million cases of illnesses in the United States every year. The WSF obtained using a Clevenger‐type apparatus was further divided into two equal parts, one each for in‐vivo toxicity evaluation and quality assessments, respectively. The proportions of major phenylpropanoid identified as meta‐eugenol in the WSF were found in the range of 42.8‐57.9%, which was substantially in higher proportion as compared to essential oil (20.9‐23.0%). Based on sub‐acute oral toxicity data, WSF has not shown any adverse effect with levels as high as 500 µL/25g body weight in Swiss albino mice. Besides, the WSF also exhibited a maximum reduction in bacterial load in mice infected with Salmonella Typhimurium in a dose‐dependent manner. We have shown the biological potential of basil water‐soluble fraction as an effective bacterial load‐suppressing agent for the prevention of Salmonella infections in animal model.