Energy requirements continue to rise despite depletion of fossil energy. Therefore, environmentally friendly renewable alternative energy is needed including bioethanol. Microbial assistance is usually required to convert the sugar content from sugary content (coconut sap) into ethanol. This study was aimed to determine the performance of several microbes and the optimum fermentation time for bioethanol production from coconut sap. Randomized block design with 2 factors was used, where the 1st factor was the type of microbes consisting of isolates R5I3, IS258, and S. cerevisiae ATCC 9763, and the 2nd factor was the fermentation time, namely: 0, 1, 2, 3, and 4-days. The results showed that the best isolate for producing bioethanol from coconut sap was isolate IS258, followed by isolate R5I3 with the least was S. cerevisiae ATCC 9763. Isolate IS258, R5I3, and S. cerevisiae ATCC 9763 was able to produce ethanol of 6.18±0.45%, 5.39±0.21% v/v, and 4.65±0.83% v/v, respectively and in 3, 3, and 4 days of fermentation, respectively. In the process of bioethanol production based on coconut sap, IS 258 isolates is potentially be used in the production process in the future.