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, KH2PO4, NaCl, MgSO4 7H2O and Tween-80 in shake flask experiments. Initial screening using a Plackett.Burman statistical design identified four components, i.e., molasses, SSC, KH2PO4 and MgSO4 7H20 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; KH2PO4 = &1.484 and MgSO4 7H2O = &0.072.