Sodium caseinate fermented of Lactobacilli were studied for their antimicrobial activity. Out of the total 34 standard NCDC, 12 Lactobacillus cultures were selected on the basis of maximum proteolytic activity. Proteolytic property was used to study their antimicrobial activity against pathogenic and contaminating organism. This Lactobacillus was assessed for probiotic attributes. Proteolytic and probiotic strains are selected for studying antimicrobial activity using caseinate fermented. Cell free supernatant of Lactobacillus was tested for antimicrobial activity. Sodium caseinate (2.5%) was prepared for fermentation; concentrated 10 fold at 60°C was tested for antimicrobial activity against gram positive and gram negative bacteria, yeast and mold cultures. Lb. fermentum 141 had maximum antimicrobial activity as compared to other Lactobacillus and selected for further studies. Minimum inhibitory concentration of Lb 141 fermentate (MIC) against bacterial cultures was 100 µg / ml whereas 120 and 150 µg / ml for mold and yeast respectively. Sodium caseinate fermentate of Lb 141 showed bactericidal and fungicidal activity against a broad spectrum of microorganisms. Antimicrobial fermentate of Lb 141 may act as a natural preservative in milk food products against undesirable bacteria as well as yeast and molds.