Sclerotium rolfsii is a soil-borne fungal pathogen that causes southern blight disease characterized by the wilting and yellowing of leaves on a wide range of agricultural and horticultural crops. Recent studies have shown an increase in the incidence of southern blight in tomato plants. During colonization of host tissue, it produces a considerable mass of mycelia on the plant’s surface leading to tissue decay and subsequent production of sclerotia. S. rolfsii is known to produce sclerotial exudates which are thought to help the fungus survive. Analyzing the sclerotial extract (SCE) for anti-microbial activity may lead to the development of a biopesticide. We evaluated 5 different fungal culture media for sclerotia production in the laboratory and assayed for antifungal and antibacterial activities on various plant pathogens. Furthermore, tomato calli initiated from hypocotyls were treated with SCE and grown on solid MS media containing 3 mg/L or 10mg/L kinetin at 22±2°C and 16 h/8 h light/dark photoperiod for four weeks. The controls were treated with sterile distilled deionized water. SCE effect on tomato callus was assessed by callus fresh weight, tetrazolium chloride staining and electrolyte conductivity. Sclerotia production was greater in oatmeal agar and potato dextrose agar than V8 juice, cornmeal agar and 2% water agar. Although sclerotial extracts did not affect the growth of Alternaria solani, they reduced the growth of Leptosphaeria maculans and completely inhibited the growth of Erwinia amylovora and Xanthomonas campestris. Callus growth was prolific in media containing 3mg/L or 10mg/L kinetin but was significantly repressed by SCE especially in media containing 3mg/L kinetin. About 50% of SCE treated callus tissue became necrotic 10 days after treatment. Our results also indicate that SCE of S. rolfsii have considerable antimicrobial activity against some plant pathogenic fungi and bacteria, and significantly inhibit tomato callus growth especially at low kinetin concentration.