Root rot of alfalfa caused by Rhizoctonia solani is considered one of the most important diseases, which annually occurred in many plants and causes great losses in yield production. So the present study aimed to find the best strategy to control or at least reduce disease severity. The Results showed that the fungal isolates were identified by using the morphological features of mycelia as R. solani. Effect of certain antagonistic bio-agents fungi to inhibit the growth of R. solani has been studied under laboratory. In general isolate No. 2002, followed by 2008 and 2004, which recorded (60.5, 47.2and 33.1%), respectively In this respect, the highest inhibition of R. solani growth was occurred by isolate No.5001, followed by 5007 and 5009, 5002, which recorded (54.56, 449.39, 46.26 and 46.26 %), respectively. Eight bacterial strains were tested against growth of Rhizoctonia sp was investigated in vitro .demonstrated that all tested bacteria significantly inhibited growth of Rhizoctonia sp in vitro.