Damping-off caused by Pythium aphanidermatum, as one of the most infamous plant diseases, is considered as a significant disease that causes severe damages in greenhouse and field crops in a vast range of hosts especially vegetables. The application of chemical fungicides as a common method to control this disease poses negative side effects on humans and the environmental components. Actinobacteria, especially members of the genus Streptomyces, are fascinating biocontrol agents and plant growth-promoting rhizobacteria, which exhibit safer alternative managements to control this disease. The present study aims to explore for bioactives soil Streptomyces isolates that are able to control P. aphanidermatum, which is the causal agent of damping-off in tomato. Out of a total of 116 actinobacteria isolates collected from the soil, 53 have showed an antagonistic activity against P. aphanidermatum, as deduced through in vitro dual cultures. Based on in vitro Petri plate seedling-bioassays (IPSB), from the 53 tested isolates in dual cultures, two isolates coded as H2 and H3, considered as the most bioactive agents, were selected to assess their biocontrol performances against P. aphanidermatum in the Sandwich bed-mix technique in greenhouse experiments. In vivo greenhouse statistical studies were performed to compare seven treatments using completely randomized design experiments. Metalaxyl fungicide was applied as the chemical-control treatment. To evaluate biocontrol efficiencies of the two Streptomyces symbionts, disease incidence recorded throughout the course of experiment and criteria of number of leaves, length of the plants, plant fresh and dry weights, were measured at the end of experiment and analyzed statistically. The resulted disease incidences for all treatments indicated that the two Streptomyces strains had PGPR activity, and they were effective in decreasing disease incidence and improving plant performances regarding number of leaves, height, and plant fresh and dry weights. Based on the phylogenetic analysis of the partial sequences of the 16S rRNA gene, the strain H2 revealed a close relation to six Streptomyces species, namely S. badius, S. caviscabies, S. globisporus, S. parvus, S. sindenensis, and S. griseoplanus, with 99.9% similarity. The strain H3 also indicated a close relation of the same similarity to two species, namely S. flavogriseus and S. pratensis. In overall, collected data dictated that the two bioactive root symbiont Streptomyces strains effectively controlled the damping-off disease caused by P. aphanidermatum.