This study aims to screen new LAB from Algerian cow’s milk to assess their probiotic properties. Molecular identification and MALDI-TOF mass spectrometry methods were used to identify the LAB isolates. The probiotic potential of isolates was determined with in vitro tests of survival to gastrointestinal conditions (pH 2, 0.3% pepsin, 0.5% bile salts, 0.1% trypsin, and 0.1% pancreatic amylase) and antimicrobial and antioxidant activities. Eight isolates were identified as Lactiplantibacillus plantarum (100%) and one isolate as Lacticaseibacillus rhamnosus (95.75%). The MALDI-TOF MS analysis of the isolates confirms that the strains belong to the group of lactobacilli bacteria, particularly Lactiplantibacillus plantarum. The high survival rate reflects a good strain tolerance to the in vitro host simulated gastrointestinal conditions. All bacteria exhibit an antibacterial activity strain with inhibition zone diameters ranging from 4.9 mm against Aspergillus niger ATCC 106404 to 17.47 mm against Candida albicans ATCC 10231. The antioxidant activity with the highest DPPH scavenging activity (92.15%) was obtained with the LbN09 strain. In light of these results, some of the strains isolated from raw milk of the local Algerian breed cows show promising probiotic properties, giving them a possible use in preserving food from microbial spoilage and oxidation during storage.