Toxicity of the fungicide Flutolanil was in vitro tested against 20 isolates of Macrophomina phaseolina and cotton seedlings of ten commercial cotton cultivars. The isolates were recovered from roots of cotton plants obtained from different cotton-growing areas in Egypt. Most of the tested isolates were sensitive to Flutolanil; however, they varied in sensitivity. Twenty-five percent of the isolates were highly sensitive where IC50 ranged from < 1 to 5.1 µg/ml, 20% of the isolates were sensitive where IC50 ranged from 15 to 30 µg/ml, 45% of the isolates were moderately sensitive where IC50 ranged from 46 to 58.5 µg/ml, and 10% of the isolates were not much sensitive (tolerant) where IC50 was > 100 µg/ml. Flutolanil was very safe on both shoots and roots of the tested cultivars (IC50 > 100 µg/ml). Treating cotton seeds with Flutolanil resulted in highly significant (P < 0.01) reductions in pathogenicity of 18 isolates and a significant reduction (P < 0.05) in pathogenicity of isolate M29. M1 was the only isolate, which was insensitive to the application of Flutolanil. In vivo toxicity to Flutolanil was not correlated with its in vitro toxicity. However, a highly significant correlation (r = 0.60, P < 0.01) was observed between pathogenicity of isolates and the in vivo toxicity of the fungicide.