This study explores the biosynthesis of bimetallic nanoparticles using
Camellia sinensis
extract and their effects on the gut symbiont
Lactobacillus gasseri
. Ag/Au nanoparticles (NPs) with Ag:Au molar ratios of 4:1, 1:1, and 1:4 were characterized by uniform alloyed morphology, crystalline (111) planes, and particle sizes of 18.5–32.8 nm. Antibacterial assays revealed that Ag:Au molar ratios of 4:1 reduced
L. gasseri
growth by 92% and caused a 2.9-fold increase in malondialdehyde and a 46% decrease in glutathione, indicating pronounced oxidative stress. Metabolomic profiling showed significantly suppressed key metabolic products. Short-chain fatty acid (SCFA) analysis revealed a 73.4% drop in butyrate, accompanied by a 2.8-fold rise in extracellular lipopolysaccharides (LPS), indicating increased inflammatory potential. Microbial community analysis via 16S rDNA sequencing demonstrated that decreased Shannon diversity from 3.45 to 2.11 and reduced
Lactobacillus
abundance from 42.3% to 11.4%, while
Escherichia
and
Streptococcus
populations expanded. In contrast, Ag/Au nanoparticles with Ag:Au molar ratios of 1:4 caused minimal shifts in both microbial composition and function. These findings demonstrate that the Ag content critically determines nanoparticle-microbiota interactions, with silver-rich formulations disrupting metabolic activity, ecological balance, and redox homeostasis. Gold-rich nanoparticles NPs, by contrast, exhibit superior compatibility with probiotic taxa and preserve gut-relevant metabolic functions. This highlights the potential of green-synthesized, Au-dominant nanomaterials in microbiome-preserving biomedical and nutritional applications.