E.coli is believed to be one of the most main bacteria in the population which constitutes the normal microbial flora. E.coli strains have been found in the gastrointestinal tract of humans, poultry and other animals as harmless bacteria, but despite the harmless nature of its existence, E.coli can be opportunistic if the host immunity status wasn't intact. In this research work, evaluation, and investigation of the anti-bacterial activity of silver nanoparticles which synthesized by a green chemistry methodology through utilizing Aspergillus niger was carried out. The isolation of E.coli bacteria has been completed through sampling of different regions of the chickens, such as the cloacal region, intestinal fluid, and faecal material. For identification of E.coli, biochemical examinations, like catalase, oxidase, urease, triple sugar iron test, citrate utilization test, and indole test were applied. Silver nanoparticles characterization was accomplished through using different physicochemical techniques, namely, Fourier transformed infrared (FT-IR) which has been used to obtain the distinctive molecular fingerprint of silver nanoparticles through the formation of the infrared absorption spectrum, Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to display the morphology of the nanoparticles. Agar well diffusion method has been executed to evaluate how effective silver nanoparticles are as anti-bacterial agent against E.coli strains by means of inhibition zone and MIC values. The values of the inhibition zone and MIC obtained were 14 mm and 39.06 µg/ml respectively.