The increasing demand of consumers who look for natural safe products and associated health risks of chemically treated and refined goods food products led to the introduction of alternative technologies for Preservation and maintenance of dietary freshness. One of such Preservation technology requires the use of Lactobacilli as starter culture for the preservation of food matrixes planned. The present study was designed to evaluate sweet whey low-cost by- product of dairy manufacture as a substrate for the selection of Lactobacilli with proven antibacterial activity to be used as biopreservation for fermented dairy product. Human population has used dairy products, including milk, as a source for Lactic acid bacteria (LAB). The positive impact of the given bacteria as a diet supplement has been of concern to researchers. The target of this study is to examine Lactobacillus species isolated from dairy products i.e. raw milk alongside cheese, with potential activities. Using the Sweet whey as growth medium for Lactic acid bacteria. Also, tested of antibacterial activities. Sweet whey (sw) proved to be suitable medium alternative to the expensive commercial De Man-Regosa-Sharp medium for the growth and production of antibacterial substances by isolated lactic acid bacteria isolates. Therefore, was used throughout the present work. A number of 32 isolates were obtained from raw cow milk (16 isolates), goat milk (9 isolates) and cottage cheese (7 isolates) using the specific De Man-RegosaSharp medium (MRS) for the isolation of LAB, these isolates were identified up to genus as strains of Lactobacillus spp. Among 32 isolates of Lactobacillus spp., there are twenty four isolates antagonized of the all seven tested pathogenic bacteria. The screened Lactobacillus spp. isolates were characterized and selected the best bio agent isolate against pathogens was identified using 16s DNA gene to Lactobacillus brevis. This strain was resistant to NaCl (2-6.5 %), produces dioxide carbon (CO2) and showed good growth in different temperature (10°, 15, 45°C) and fermented of many sugars. As per the outcomes shown herein, the strain in question was thought to be antimicrobial bacterium produced a variety of metabolites, including butyric, formic, lactic, citric, and sorbic acid. The given set of activities adds to microbiological safety by helping control microorganism growth, and inhibiting pathogenic bacteria by using the lower-cost sweet whey. Therefore, more detailed work on isolating and characterizing antibacterial bacteria from the locally produces dairy items, and augmenting their growth could be needed for the creation of biopreservative foods.