In this study, a wide survey was conducted along Saudi Arabia. One hundred soil samples were collected from different 18 governorates representing different climatic conditions. Five hundred and seventy strains of actinomycetes were isolated from the collected soil samples. Among them, 225 were found to be antagonistic to the pathogenic fungus (Fusarium oxysporum f. sp. lycopersici) with varying degrees. Only one isolate designated E44G, had the strongest antagonistic activity against the tested fungus. The taxonomic status of this isolate was established using Phenotypic and molecular methods. The optimum nutritional and environmental conditions were studied to produce the maximum yield of antifungal activity. The highest antifungal activity was obtained at 2nd day of incubation, 7.5 pH, and 30 °C. Glucose was the best carbon source and yeast extract was the best nitrogen source. In the present study, we report on a potential Streptomyces strain which has antifungal and antibacterial activities and holds the potential for use in studies of bioactive compounds as well as for possible use in biological control of fungal and bacterial diseases of important crops.