This study was to determine the spoilage microbiota of palm wine stored at room temperature in order to define appropriate strategies for shelf-life extension. For this purpose, during the storage of palm wine samples, yeast, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) counts were performed. Then, species were identified by PCR-RFLP followed by sequencing and the overall bacterial community was determined by NGS (Next Generation Sequencing). The results showed that yeast, LAB and AAB counts decreased in the palm wines collected from producers after 24-48 h, while they remained constant in samples collected from resellers. Furthermore, the stored palm wine microbiota was composed of several yeast species namely Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Candida parapsilosis and Brettanomyces bruxellensis. LAB species were Enterococcus durans, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Fructilactobacillus durionis, Lactobacillus parafarraginis and Lactiplantibacillus pentosus. For AAB, the species were Acetobacter oryzoeni and Acetobacter pasteurianus. The species whose proportions increased steadily during storage were B. bruxellensis, C. parapsilosis, Lc. paracasei, L. fermentum and A. oryzoeni. The NGS method confirmed that Lactobacillus was predominant during storage, followed by Zymomonas and Acetobacter. Knowledge of the microorganisms present during the spoilage of palm wine and their dynamics will enable the development of preservation methods specifically targeting the microorganisms identified.