Abstract: In vitro studies were conducted on the effect of temperature, pH levels, culture media, carbon and nitrogen sources on mycelial growth of Sclerotium rolfsii Sacc. Growth of S. rolfsii was maximum at 25⁰C after 7 days of inoculation, which was reduced significantly below 20⁰C and above 35⁰C. All the tested pH levels (5 to 8) were found equally suitable for growth of fungus. This fungus grew best on cornmeal agar medium among the culture media that were tried. All the carbon sources were found to be the best while peptone was the best among the nitrogen sources.