Biotechnological studies on production of bio-surfactants by actinomycetes strains that were isolated from Egyptian soil were carried out using some diesel oil waste samples. Two actinomycetes strains were found to give bio-surfactant production up to 40 - % when tested with emulsification test. Effect of carbon and nitrogen sources on bio-surfactant production were tested using urea, ammonium nitrate, ammonium chloride and potassium nitrate. Best results for bio-surfactant production were observed with urea and ammonium nitrate as the main nitrogen source in the biological medium. Best results for bio-surfactant production were observed when using Bushnell Hass medium supplement with % hydrocarbon waste. Ammonium sulfate precipitated samples were further applied on Sephadex LH-20 column to obtain purified products. Results obtained from isolated strains show optimistic results for a successful bioremediation of diesel oil wastes for production of commercial active bio-surfactants that could be used in both industrial and pharmaceutical fields.