The current study aimed to improve riboflavin production by native bacterial isolates and medium components. Bacterial species Bacillus subtilis ASU8 (KU559874) and Bacillus tequilensis ASU9 (KU559876) showed high potentiality for riboflavin production. The optimal growth and riboflavin production were achieved after 48 h. The impact of glycine addation to the basal medium was explored by applying different concentrations (0.1-3 g/l). The addition of glycine was effective and the influencing concentration was 1 g/l giving 144.7 and184.2 mg/l yields of riboflavin for Bacillus subtilis ASU8 and Bacillus tequilensis ASU9, respectively. Medium constitu- ents were also optimized for high riboflavin yield using central composite design (CCD) of response surface methodology (RSM) to optimize the significant values and to study the mutual interactions between these constituents. The analysis revealed that optimal medium concentrations for maximum production of riboflavin by Bacillus subtilis (273.82 mg/l) and Bacillus tequilensis (288.33 mg/l) were (g/l): glucose 60, 40; NaNO 3 3, 5; KH 2 PO 4 2.5, 1.5; K 2 HPO 4 1, 0.5 and MgSO 4 .7H 2 0.1, 0.5 for Bacillus subtilis and Bacillus tequilensis , respectively. Analysis of variance results showed that the model was significant and an R2 value of 98.06% ( Bacillus sub- tilis ), 99.34% ( Bacillus tequilensis ) demonstrated that the experimental results were fitted well with predicted values. Optimization of medium conditions using RSM experimental design is an efficient approach for the production of riboflavin through microbial fermentation by Bacillus subtilis and Bacillus tequilensis could be utilized in industrial application