Lafun is a fermented cassava food product consumed in parts of West Africa. In the present work the microorganisms (aerobic bacteria (AB), lactic acid bacteria (LAB) and yeasts) associated with the fermentation of Lafun under traditional conditions have for the first time been studied using a combination of pheno- and genotypic methods. During Lafun fermentation the AB count ranged from 6-7 log(10) CFU/g at the beginning to 9 log(10) CFU/g at the end. Similarly, the number of LAB increased from 5 log(10) CFU/g to 9 log(10) CFU/g during the process while the yeast load increased from 3 log(10) CFU/g at the onset of the fermentation to 5-6 log(10) CFU/g at the end of the fermentation. A total of 168 isolates (31 AB, 88 LAB, and 49 yeasts) were isolated and identified by means of phenotypic tests, PCR-based methods and 16S rRNA gene sequencing. The aerobic bacteria were mostly identified as belonging to the Bacillus cereus group (71%). The B. cereus isolates lacked the genetic determinant specific for cereulide producers but harboured several genes encoding the heat-labile toxins hemolysin BL and nonhemolytic enterotoxin as detected by PCR. The other aerobic bacteria isolated were Gram negative and identified as Klebsiella pneumoniae and Pantoea agglomerans. The dominant LAB were identified as Lactobacillus fermentum (42% of LAB isolates) followed by Lactobacillus plantarum (30%) and Weissella confusa (18%). Seven isolates remained unidentified and constitute probably a novel LAB species. The predominant yeast species associated with Lafun fermentation were Saccharomyces cerevisiae (22% of yeast isolates), Pichia scutulata (20%), Kluyveromyces marxianus (18%), Hanseniaspora guilliermondii (12%), Pichia rhodanensis (8%) and Candida glabrata (8%) as well as Pichia kudriavzevii, Candida tropicalis and Trichosporon asahii at lower incidence (<5% each).