All the microorganisms produce primary and secondary metabolites which have various industrial applications. Metabolites such as antibiotics are mainly produced by the filamentous micro- organisms such as bacteria and fungi. The rhizosphere is a narrow zone surrounding and influenced by plant roots and also a hot spot for several organisms. Organisms found in vicinity of roots include bacteria, fungi, oomycetes, nematodes, protozoa, algae, viruses, archaea, and arthropods. Microorganisms in rhizosphere are different from those residing in the non-rhizosphere surrounding soil, both in numbers of cells and the variety of strains. Rhizosphere microorganisms have been recognized as an important source of a variety of structurally unique and active secondary metabolites. In present study, 6 fungi were isolated form rhizosphere of medicinal plant Azadirachta indica (Neem). Out of 6 isolates, one isolate was selected and identified (Aspergillus niger) as showed maximum antimicrobial activity against test microbe E. coli. Fermentation conditions were optimized such as temperature (25°C), pH (5), carbon source (glucose) and nitrogen source (ammonium nitrate) for maximum production of antimicrobial compound.