In Burkina Faso, the Palmyra Palm Borassus aethiopum Mart. grows wild and gives natural stands in Central-Eastern and Eastern regions. The sap collected traditionally ferments spontaneously and is a rich medium that allows the growth of different microorganisms. This study aimed to identify yeasts and lactic acid bacteria (LAB) isolated from Borassus aethiopum Mart. fresh and fermented sap in Burkina Faso. A total of ninety strains including thirty LAB and sixty yeasts were isolated in the fresh and fermented sap. The isolates were characterized using standard biochemical method and sequencing of the V1 to V6 region of 16S rDNA of LAB and 28S rDNA of yeasts. The neighbour-joining method was used for the construction of phylogenetic tree with MEGA X software. After biochemical characterization and sequencing of the V1 to V6 region of 16S rDNA, twenty LAB strains (67%) were identified as Leuconostoc mesenteroides, seven (23%) as Enterococcus sp. and three (10%) as Enterococcus gilvus. Sequencing of the yeast 28S rDNA showed that 63% of the strains were Saccharomyces cerevisiae and 37% Pichia kudriavzevii. Leuconostoc mesenteroides and Saccharomyces cerevisiae are commonly isolated from several palm sap or wine of palm trees, but Enterococcus sp. and Pichia kudriavzevii are not commonly detected in palm wine. The LAB species Enterococcus gilvus identified in our study has not yet been isolated previously in palm wine. The yeasts and LAB isolated from Borassus aethiopum sap are the main microorganisms responsible for sap fermentation and could be used for several biotechnological applications