This study was intended to study the biological activity of B. subtilis and silver nanoparticles against MDR E. coli isolated from different infections. in the province of Najaf. one hundred fifty_ five samples and inquiries were carried out between (October, 2021 to January, 2022). were collected from patients with different infection from two separate hospitals Al-Hakam Hospital and Al- Zahraa Hospital as well as AL-Aman center for research AL-Najaf Governorate, Isolates were identified based on the morphological and microscopic examination, and VITEC. Antibiotics susceptibility test by modified Kirby - Bauer disc diffusion method the results showed (58) isolates, from, E. col resistance against 22 commonly used antibacterial agents. the capacity of some E. coli isolates to biofilm formation was detected by phenotypic method which included Congo Red Agar Method (CRA), from the 58 (100%) isolates of E. coli 48 (82.8%) were biofilm producers when that appearance of black dry crystalline colonies on the CRA plates and 10 (17.2 %) were non - biofilm producers when the colonies of E. coli remained pink or red colored. Molecular study was detected the antibiotic resistance genes( gyrA), (Sul1), (Ant(2")-1a), (ermB), (blaSHV) and (qnrA) in E. coli isolates finally, the isolates were subjected to experiments with two Treatments B. subtilis and silver nanoparticles The results showed that, inhibition zone of E. coli increased progressively with increase the silver nanoparticles and B. subtilis concentrations in reaching a optimum inhibition in 400 µg/ml,0.01mg/ml respectively, also antibacterial activities of some resistance antibiotics included Amoxicillin-clavulanic acid, Levofloacxin, Gentamicin, and Trimethoprime-sulfamethaxazol exhibited that the presence of silver nanoparticles with B. subtilis together with the resistance antibiotic resulting in decrease in the ability of the antibiotics to inhibit the E. coli resulting in the occurrence of inhibition zone of E. coli and insignificant compared with the presence of silver nanoparticles alone as well as more than the case of the presence of B. subtilis alone with these antibiotic. the aim of this study is to find novel therapeutic agents.to to control some diseases, and contribute in reducing the problem of the rapid emergence of antibiotic resistant pathogens.