Date Palm sap is a fresh juice called "Legmi" widely produced and consumed in Southern Tunisia. The aim of this research was to study microbiological group's distribution present in 10 palm sap samples, and to select Lactic acid bacteria (LAB) with suitable properties for use as starter cultures in sap lactic fermentation. Microbiological analysis showed high load in fresh palm sap. Mesophilic aerobic bacteria varied from 6.07 to 8.57 log cfu/ml, coliforms ranged from 3 to 6.78 log cfu/ml, yeasts between 3 and 8.47 log cfu/ml and LAB from 5.36 to 8.47 log cfu/ml. Ten strains were chosen randomly to study the acidifying activity when growing in palm sap. These strains, tentatively identified by API 50CHL, were divided into two groups: Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus delbrueckii subsp. delbruckii. All the strains were considered as fast acidifier since pH was higher after 6 h of fermentation. The higher acidifying strains KH 3 (L. delbrueckii) and 5B 4 (L. mesenteroides), were used to inoculate sap in pure culture. During the fermentation, pH decreased from 6.94 and 6.36 for 5B4 and KH3, respectively, to about 4.0±0.1 after 24 h of fermentation for both strains. Total titrable acidity increase from 0.2 and 0.4% for 5B4 and KH3 respectively to around 3.0%. The application of these strains as starter cultures in the production of fermented palm sap could be investigated on further studies.