Ralstonia solanacearum is a soil-borne bacterial that causes a lethal wilt disease in tomatoes and other economically importan crops. The best biocontrol efficacy is obtained by selection an antagonist compatible with environmental conditions and plant Rhizosphere. selected the screening method of potential antagonist is important to achieving in biological control. Several studies have shown that in vitro antibiosis does not necessarily relate to the same effect under in vivo conditions. In this study, one hundred twenty isolates sampled from healthy tomato plant roots of rhizosphere soil and were evaluated biocontrol effects and inhibition rate under laboratory condition. Initial Screening was performed the each of two methods and effective antagonists were selected based on greenhouse studies. At first, the inhibitory ability of the isolates was measured on the Nutrient agar medium (NA) and diameter's halo of inhibition. eight isolates, which showed the maximum inhibition zones, were select for further study. Then in-vitro was evaluated of potential biocontrol isolates. the cell suspensions to an optical density of 0.1 at 600nm (OD600 = 0.1) were added to the penicillin glass containing hydrogel culture medium and used for the inoculation of sterile tomato seedlings (Falat cultivar) were cultured in hydrogel. After 8 days, the symptoms start necrosis and wilting were visible in inoculated plants. Comparison of two screening methods showed that only some of the isolates had similar antagonistic effects in both methods. After a greenhouse survey it was found that the selected isolates in the penicillin-in-glass method had specific antagonistic effects in the pots and successfully prevented plant infection. By reviewing the result it can be seen that the observations recorded in greenhouse conditions confirm the accuracy of the in vitro test results. Therefore, this method can be used to screen large number of isolates in a limited space similar to the greenhouse conditions and avoids interference of other bacteria/micro biota from soil during the infection process. This rapid and sensitive screening method for understanding the interaction between beneficial bacteria, plant pathogens in similar conditions of greenhouse was offered attractive alternatives to the conventional methods for the antibiosis the disease. More studies are under evaluation to identify isolates.