Fermented locust bean seeds are utilized for seasoning in various traditional dishes. Its production is mostly under unhygienic conditions by majorly illiterate women who care less about microbial contamination of the final product. Consumption of any contaminated food can cause health complications in humans. DNA extraction offers a distinct method for identifying organisms. This study aimed at isolating the mycoflora associated with fermented locust bean seeds collected from 3 villages in Kwara State, Nigeria using both morphological and molecular techniques. Morphologically, isolate A was yellowish – green, B was black; C was initially white, but turned to brown later, while D was cottony and white in colour. DNA of each of the isolates was extracted using Zymo Fungal/Bacteria DNA Miniprep Kit. PCR amplification of the ITS regions of the isolates was carried out using primer pair; ITS1 and ITS4. The products were then sequenced and the results after BLAST search revealed that Isolate A is Aspergillus flavus, B is A. niger, C is Rhizopus arrhizus and D is Mucor indicus. The fermented locust bean seeds from Madi village had the highest fungal count (7.2 X 103 cfu/ml), while that from Ogundele village had the lowest (1.4 X 103 cfu/ml). The presence of Aspergillus niger and A. flavus (mycotoxigenic fungi) in this study poses a significant public health risk. Therefore, it is important to encourage the villagers involved in fermented locust bean production and storage in these villages to be more hygienic.