The effects of cultural conditions on the production of anti-MRSA activity of the endophytic fungal isolate, Lasiodiplodia pseudotheobromae IBRL OS-64 isolated from the leaf of a local medicinal herb, Ocimum sanctum Linn. was studied. The highest anti-MRSA activity of 50.0±0.1 U/mL and 1.46±0.1 g/L of fungal growth were obtained after incorporating all the improved cultural conditions which consisted of yeast extract sucrose broth supplemented with host plant extract, with initial medium pH of 6, 2 mycelial plugs of 4 days old seed culture, cultivation temperature of 30°C and cultivated for 16 days under static conditions without the presence of light. After improvement of cultural conditions, a significant increment of anti-MRSA activity of about 57.57% (2.4 folds) and a slight increment of fungal growth of about 8.15% (1.1 folds) were obtained compared to the cultural condition before improvement. Indeed the improvement of cultural conditions greatly affected the anti-MRSA activity and growth production by L. pseudotheobromae IBRL OS-64 isolate.