Lactic acid bacteria (LAB), which are the most frequently used probiotics in foods, confer health benefits such as antimicrobial activity, immune stimulation, and anticancer activity. Fermented palm sap is a potential source of LAB. This study aimed to evaluate in vitro antimicrobial and probiotic properties of LAB isolated from traditional fermented palm sap in Thailand. Among 40 isolated LAB species, 10 were preliminarily selected for their antimicrobial activity. These 10 isolates were identified and confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA sequencing as Lactobacillus paracasei (8/10), Lactobacillus fermentum (1/10), and Lactobacillus brevis (1/10). They were evaluated for probiotic characteristics and antimicrobial activities against pathogens. These isolates were tolerant toward simulated gastrointestinal tract conditions, including low pH, pepsin, pancreatin, and bile salts. The 10 isolates retained strong auto-aggregation and cell surface hydrophobicity, and they adhered tightly to human intestinal epithelial cells. The isolates were susceptible to ampicillin, erythromycin, clindamycin, tetracycline, and chloramphenicol but resistant to vancomycin, kanamycin, and streptomycin. Moreover, all isolates exhibited no hemolytic activity. All isolates exhibited good antibacterial activity against nine pathogenic bacteria. Thus, these 10 Lactobacillus isolates from fermented palm sap are promising potential candidates for use as probiotics in functional fermented foods and pharmaceutical products.