This work is an attempt to overcome antimicrobial resistance problem which dispersed worldwide inparticular developing countries due to misuse of antibiotics. Actinobacteria were isolated and screenedagainst selected resistant Gram-negative bacteria to detect the powerful antibacterial activity.Identification of the most potent actinobacterial isolate has been carried out using classical and geneticalmethods. Antibacterial compound has been extracted, purified and characterized using accurate and morespecific techniques and instruments. Among forty actinobacterial isolates, only twenty-two isolates couldinhibit the growth of Gram-negative bacteria. The most potent isolate Eg-7 was identified as S.griseorubens, which has a typical 16S rRNA gene. The antibacterial compound was extracted using ethylacetate, and separated by High Performance Liquid Chromatography using methanol and water as amobile phase. Five active peaks were displayed and retained in the range 40 – 45 min, but the last threepeaks were retained at 41.90, 43.43, and 44.54 min, respectively. The crude extract was analyzed byliquid chromatography mass spectrum, where the active peak was displayed at 721.325 m/z. Theantibacterial compound was purified using flash column chromatography and gel filtration columnchromatography. The active fraction was analyzed by Infra-Red spectrum, where a broad absorption at3338 cm-1 was displayed. Molecular formula of an antibacterial compound was determined by massspectrum as C35H26N6O4. Nuclear magnetic resonance analysis was carried out for an antibacterialcompound. These results suggest that a new antibacterial compound that similar quinolone could beproduced by S. griseorubens and exhibited a higher activity against Gram-negative bacteria.