Enset (Ensete ventricosum) is the basis of the staple food consumed by about 20% of the Ethiopian population. Kocho is one of the food products generated from enset by spontaneous fermentation of decorticated and pulverized pseudostem and corm sections. We isolated culturable microbes associated with kocho from different stages of fermentation. Twelve yeast species, six lactic acid bacteria (LABs) species and eleven species of aerobic bacteria were identified by sequencing ITS/D1D2 regions of 26S rDNA of yeasts and 16S rDNA of bacteria, respectively. More yeast species were identified in fresh (fermented for 2–5 days) kocho, compared to long-term (7–12 months) fermented kocho, while we observed an opposite trend for LABs. In fresh kocho, the most frequently isolated yeast species were Pichia exigua, Galactomyces geotrichum, and Pichia fermentans. From mid-term (3–4 months) kocho most frequently Candida cabralensis, G. geotrichum, and Candida ethanolica were isolated. In the long-term fermentations, the most frequently isolated yeast was Saturnispora silva. Lactobacillus plantarum was the most frequently isolated LAB in both fresh and mid-term kocho. In long-term fermented kocho, Acetobacter pasteurianus and L. plantarum were most frequently isolated. L. plantarum was consistently isolated from all the three stages of fermentation. Aerobic bacteria in fresh kocho were mostly gram-negative, with Raoultella planticola and Pantoea agglomerans being the most frequently isolated species. In long-term fermented kocho, mainly gram-positive, spore-forming bacteria of the genus Bacillus were found, among them also species of the Bacillus cereus group, Bacillus anthracis and Bacillus thurigiensis.