Extended-spectrum β-lactamase (ESβL) producing E. coli pose a significant medical challenge. It has spread worldwide, making it as the source of a variety of urinary tract and wound infections. Recently, nanosilver has been widely utilized in the medicinal applications. We aimed at fabricating silver nanoparticles (AgNPs) and observing how they affected the ESβL-producing E. coli isolated from different human anatomical regions. Under optimal circumstances, AgNPs were biosynthesized using Bacillus thuringiensis isolated from raw milk and they were wholly characterized. The AgNPs were occurred to be crystalline and have a stable structure. AgNPs are hexagonal and spherical in form with no considerable agglomeration, according to TEM-results. The particle diameters varied between 13.2 and 36.8 nm. AgNPs and AgNPs-cotton clothing for wound treatment and self-sterilizing coats were displayed to have an antimicrobial action versus ESβL-producing E. coli. AgNPs, and their application i.e., AgNPs-wound dressing, shown a considerable antibacterial efficiency against ESL-producing E. coli. The minimum inhibitory concentrations (MIC) of AgNPs against ESβL-producing E. coli ranged from 7 to 9 μg mL−1, while the MIC of AgNPs with ampicillin was 1.25–4 g mL−1. This is mostly due to the AgNPs plus ampicillin have a synergistic efficiency, increasing the antibacterial effectiveness of AgNPs by 2–4 times. Our findings implied that the generated AgNPs might be applied as a nano-drug individually or combined with ampicillin against ESβL-producing E. coli.