This study focuses on isolation, purification, identification, and evaluation of bioactive substances produced by rare actinomycetes. Various soil samples from Egypt were examined and the most potent actinomycetes isolate had been identified as Actinokineospora fastidiosa nova strain NAD R4 using phenotypic and genotypic characteristics, following the suggested Bergey's Manual International Keys for actinomycetes identification. The isolate exhibited remarkable bioactivities including antimicrobial, bioherbicidal, biofertilizer properties and the synthesis of tellurium and silver nanoparticles. Optimal conditions for biomass productivity and antimicrobial activity were achieved using sterilized starch-nitrate liquid medium, incubated at 35 °C with a pH of 5, supplemented with starch (3.0 g /100 ml) as the carbon source and L-asparagine (0.25g/100 ml) as the nitrogen source. Incubation has been going on for 7 days with speed of 150 rpm. A solvent containing ethyl acetate was used to extract the antimicrobial compound. Purification using TLC and HPLC, exhibited a potent antimicrobial activity against selected microorganisms. The purified compound was characterized through UV, IR, HNMR, and LC-Mass analysis and identified as a new derivative of izumiphenazine C, with the chemical formula C20H15N3O3. MIC and MPC of the compound were determined against different tested bacteria. The compound showed low cytotoxicity on the normal cell line of human embryonic kidney (HEK-293), while displaying high toxicity against the human liver cancer cell line (HepG2).