African locust bean (Parkia biglobosa) cotyledon is fermented in most countries of West Africa to produce a soup condiment, known as 'iru' in Yoruba language, or 'dawadawa' in the predominant Hausa language. Iru is rich in minerals and serves as a source of protein supplement in the diet of poor families. Riboflavin (Vitamin B 2 ) is an essential component of basic cellular metabolism but its daily requirement is not met in Nigeria particularly among the rural dwellers. Therefore, the provision of a riboflavin enriched iru will help to eradicate problems encountered from riboflavin deficient diet. Iru was purchased from three different markets in Ibadan, Oyo State, Nigeria. From the iru, microorganisms were isolated, characterised, screened for riboflavin production and co-cultured for the production of riboflavin enriched iru. Sixty-three bacteria were isolated and identified as Micrococcus varians (9), Staphylococcus species (27), Bacillus species (24) and Micrococcus luteus (3). Bacillus subtilis IR50 produced highest riboflavin 25.77 mg/L, followed by Staphylococcus spp. strain IR26 23.37 mg/L, while M. varians IR49 had the least riboflavin production 6.35 mg/L. Mixed culture of B. subtilis IR50 and Bacillus licheniformis IR28 produced the highest riboflavin of 4.5 mg/L, Staphylococcus aureus IR06 and B. subtilis IR50 produced 2.3 mg/L, while B. subtilis IR50 produced 1.5 mg/L when used singly. The result shows that B. subtilis IR50 have the potential to increase the riboflavin content of iru and therefore will contribute to bioenrichment technology.