Abstract: This study aimed to reduce post-harvest losses of pineapple local variety egbenana by the transformation of juice into vinegar through biotechnological process.Vinegar was produced through two successive fermentations: alcoholic and acetic fermentations. The alcohol fermentation was carried out at 30°C using yeast. Biomass, pH and Brix were evaluated daily during the fermentation. Acetic fermentation was carried out at 30°C using an acetic bacteria strain isolated from pineapple wine previously exposed to ambient temperature (28°C) for 5 days. Biomass, pH and acid levels were monitored each 2 days. The performance of acetic bacteria isolated was also assessed by studying their glucose and ethanol tolerance. The study allowed the isolation of yeast coded Saccharomyces cerevisiae (LAS01) and an acetic bacteria coded Acetobacter sp. (ASV03) both occurring in the pineapple juice. The monitoring of successive fermentations indicated that the pineapple juice with sugar concentration of 20 Brix, seeded with 106 cells of Saccharomyces cerevisiae (LAS01) for alcoholic fermentation for 4 days and afterwards seeded with 106 cells of Acetobacter sp. resulted in 4.5 acetic degree vinegar at Brix 5.3% and pH 2.8 for 23 to 25 days. The study of glucose tolerance of the strain of Acetobacter sp. showed that the growth of acetic bacteria was important in a juice with high concentration of sugar. However, the concentration of ethanol did not effect on the acetic bacteria growth. These results enabled on one hand to improve the manufacturing technology of vinegar from fruits and on the other hand to produce a starter of yeast and acetic bacteria strains for this production.