In the search for alternative therapy for infections and other ailments, metallic nanoparticles, mainly silver nanoparticles (AgNPs) synthesized through bioengineered sources are extensively explored. Fungal bioactive compounds and their nanoparticles were reported with the potential biomedical application. A medicinal mushroom
Ganoderma lucidum
was reported as a repository of rich medicinal properties. In the current study, silver nanoparticles were synthesized using the extracts of
G. lucidum
and its antimicrobial activity was tested against drug-resistant
Escherichia coli
isolated from the catheter used for urinary tract infection (CAUTI). The GC–MS study of
G. lucidum
extracts showed the presence of ethyl acetoacetate ethylene acetal with the highest area percentage of 72.2% and retention time (RT 5873). Pyridine-3-ol is the second primary compound with a peak height of 6.44% and a retention time of 2.143. The third compound is l,4-Dioxane-2,3-diol, with an area of 8.09% and RT 5450. Butylated Hydroxy Toluene [BHT] is the fourth major compound with an area of 3.32%, and 9-Cedranone constitutes the fifth position in occupying the area percentage [1.88] and height 1.56%. Pyrrole is the sixth primary compound registering an area size of 0.96% and height 2.06%. The AgNPs synthesized using
G. lucidum
extract were in size range 23 and 58 nm as per SEM analysis and within the range wavelength 0.556–0.796 nm as per UV–Vis spectral study. FTIR Spectroscopy and X-ray diffraction analysis (XRD) were made to characterize the formed nanoparticles. The AgNPs synthesized effectively inhibited the growth of
E. coli
isolated from catheter-associated urinary tract infection and showed resistance to many drugs. The antioxidant potential of the synthesized nanoparticles assessed using DPPH radical scavenging activity, EC50 (µg/ml), and ARP data showed that the prepared nanoparticles were more potent in free radical scavenging activity than the standard quercetin. The cytotoxicity effect of Ag-NPs on breast cancer cell line- MDA-MB-231 confirmed its anticancer potential. The half-maximal inhibitory concentration (IC
50
) of Ag-NPs to inhibit 50% of the tumor was 9.2 g/mL. The synthesized GL-AgNPs was exhibited a multifocal biomedical potential.