This study aimed to investigate the effects of different doses (25, 50, 75, 100, 125, 150, 200, 250, 500, 1000 and 2000 Gy) of gamma radiation on spore germination in Penicillium chrysogenum and on the production and efficacy of penicillin in this fungus. A dose of 200 Gy of gamma radiation increased spore germination by 70.2% after 20 hours of incubation compared to control cultures. It was also found that penicillin yield increased by 95.2% at this dose of gamma radiation. The efficacy of penicillin was remarkably enhanced, resulting in a 99.6% reduction in the total count of Staphylococcus aureus treated with penicillin from irradiated P. chrysogenum compared with that of S. aureus treated with commercially available and purified penicillin from nonirradiated fungi. The results obtained highlighted the potential of this technology for production of penicillin.