The effect of Ultra-Violet (UV) irradiation and acridine orange dye on L-glutamic acid producing Bacillus subtilis and B. megaterium was investigated. The selected mutant strains and wild types of B. subtilis and B. megaterium were immobilized and used for L-glutamic acid production. The Bacillus strains were exposed to UV irradiation and treated with acridine orange dye respectively. The survived Bacillus strains were found to reduce as the exposure time and concentration of the mutagens increased in this study. Four mutants of B. subtilis and two mutants of B. megaterium were selected for L-glutamic acid production. Mutant strain of B. subtilis (MAIR4) treated with acridine orange dye yielded the highest L-glutamic acid (4.62 mg/mL) at 72 hours. L-glutamic acid production by mutant wild and strains of B. subtilis and B. megaterium immobilized on sodium alginate, Agar-agar matrix and poly urethane foam ranged from 1.65 to 4.03 mg/mL, 2.04 to 3.98 mg/ mL and 1.89 to 3.39 mg/mL, respectively with B. megaterium (MUSO17) on sodium alginate producing the highest L-glutamic acid. Sodium alginate was the best supporting matrix for the production of L-glutamic acid in this research. Immobilization of mutant strains of Bacillus megaterium (MASO17) exposed to UV irradiation using sodium alginate supported the L-glutamic acid production.