The nutritional requirements for riboflavin production by a UV-mutant of Eremothecium ashbyii UV-18-57 were optimized using indigenous agroindustrial by-products. The medium components considered include molasses as carbon source, sesame seed cake (SSC) as nitrogen source, yeast extract, KH 2 PO 4 , NaCl, MgSO 4 7H 2 O and Tween-80 in shake flask experiments. Initial screening using a Plackett-Burman statistical design identified four components, i.e., molasses, SSC, KH 2 PO 4 and MgSO 4 7H 2 0 as significantly, influencing riboflavin production (confidence levels above 80%). Response surface methodology was applied to determine the mutual interactions between these four components and optimal levels for riboflavin production. The optimal concentrations for enhanced production of riboflavin were: (g/1) molasses=30.85; SSC=39.80; KH 2 PO 4 =1.484 and MgSO 4 7H 2 O=0.072.