Abstract: Lactobacillus acidophilus comes under the genus Lactobacillus which comprises of large group of beneficial bacteria that have similar properties and all produce lactic acid as an end product of the fermentation process. Previous studies shows that L.acidophilus has its own significant importance as it improves gastrointestinal function, boosts immune system, decreases the frequency of vaginal yeast infection and helps in reducing serum cholesterol levels. The present study is directed towards isolation and identification of antagonistic L. acidophilus from different regions of Allahabad. A total of 50 curd samples were collected from different regions of Allahabad. After careful examination of morphological and biochemical characteristics, all 74 isolates of different Lactobacillus species were found to be present in which 7 strains of L. acidophilus were found. Further these 7 strains were subjected to antagonistic test against selected bacterial pathogens and results revealed that all strains of L.acidophilus were found to be good antagonistic activity against selected bacterial pathogens. This study concluded that curds contain strains of L.acidophilus which shows good antagonistic property; thus revealing that it is safe and beneficial for consumption.