Actinomycetes are the main microbial population in soil generating active secondary metabolites. However, the objectives of this research were to isolate, select a promising strain and improve the antibiotic production of actinomycetes. A prospective analytical study of several actinomycetes was performed from soil samples collected from different locations in Khartoum, Sudan. The crowded plate technique was used to isolate actinomycetes using different media. The strains were evaluated for their antimicrobial activity against certain Gram-positive, Gram-negative bacteria, yeasts, and fungi. Varied fermentation conditions such as temperature, pH, and light, agitation, aeration, and fermentation period have also been configured for the maximum production of antibiotics. From 62 isolates, 18% were active against at least one of the test organisms: Bacillus subtilis (ATCC 10400), Staphylococcus aureus (ATCC 289213), Escherichia coli (ATCC 13706), Klebsiella pneumoniae (ATCC 10031), Proteus vulgaris (ATCC 630), Pseudomonas aeruginosa (ATCC 15442), Salmonella typhimurium (ATCC 13311), Aspergillus niger (ATCC 16404), and Candida albicans (ATCC 10231). The strain (O-7) showed a very broader spectrum than other isolates. For the maximum production of antibiotics, suitable fermentation conditions were found to be as follows: Temperature 28°C, pH 7.0, Agitation 180 rpm, and fermentation duration 96 hours. It can be concluded that antimicrobial compounds formed by (O-7) isolated from the soil in Khartoum were efficient. The antibiotic generated exhibited the highest activity against various pathogens.