<b>Background and Objective:</b> Actinobacteria represent the most prominent group of microorganisms, which produce a vast number of bioactive compounds especially antibiotics. The present study investigated the antibacterial activity of some actinomycete isolates against <i>Ralstonia solanacearum</i> type 3 biovar 2 (phytopathogenic bacterium that causes tomato wilt disease and brown rot of potatoes). <b>Materials and Methods:</b> The most potent actinomycete isolates in the antibacterial activity was further identified up to species based on its phenotypic and molecular characteristics. Additionally, the most suitable carbon and nitrogen sources for increasing the antibacterial activity were also investigated. <b>Results:</b> Interestingly, <i>Streptomyces </i>isolate MSQ21 achieved the highest antibacterial activity against <i>R. solanacearum</i> with an inhibition zone of 18 mm. 16S rRNA gene analysis suggested that <i>Streptomyces </i>MSQ21 was identified as a strain of <i>S. maritimus</i> Glycerol (2.25%, w/v) and (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> (0.13%, w/v) were the most suitable carbon and nitrogen sources for increasing the antibacterial activity. <b>Conclusion:</b> It could be concluded that the maximum antibacterial activity (30mm) produced by <i>S. maritimus </i>strain MSQ21 against <i>R. solanacearum </i>could be obtained by using the modified starch nitrate medium containing (g L<sup>1</sup>): Glycerol, 25: Ammonium sulphate, 1.6: Dipotassium hydrogen phosphate, 1: Magnesium sulphate, 0.5: Sodium chloride, 0.5: Calcium carbonate, 3: Ferrous sulphate and 0.01: Distilled water up to 1 L and under the following conditions: Temperature 30°C, agitation speed 250 rpm, inoculum size 1-50 mL medium, incubation period 4 days and pH 8.5.
Keywords: Ralstonia solanacearum; Streptomyces maritimus; antibacterial activity; identification; response surface methodology.