Silver Nanoparticles Produced by Rooibos Kombucha Suppress Bacterial Biofilms and Improve Survival in Galleria mellonella Infection Model

Antibiotic resistance and biofilm-associated infections require sustainable antimicrobial platforms that combine efficacy with biocompatibility. Fermented matrices are attractive for green nanomaterial production because they provide reducing metabolites and surface-active capping compounds. Rooibos kombucha is a polyphenol-rich fermentation system with potential to serve as a biosynthetic matrix for silver nanoparticles (AgNPs). The present work aimed to develop a rooibos kombucha-enabled platform for the green biosynthesis of phytochemical-capped silver nanoparticles, AgNPs-K, and evaluate their antibacterial, antibiofilm, and in vivo activity. Rooibos kombucha was fermented for 14 days and profiled by liquid chromatography–tandem mass spectrometry (LC–MS/MS). AgNPs-K were generated using kombucha extract and AgNO3, purified, and characterized by ultraviolet–visible spectroscopy (UV–Vis), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and nanoparticle tracking analysis. Antibacterial activity against eight Gram-positive and Gram-negative reference pathogens was assessed by EUCAST-based microdilution and time-kill assays. Biofilm inhibition was measured by the crystal violet assay. In vivo toxicity and therapeutic efficacy were evaluated in Galleria mellonella larvae. AgNP formation was confirmed by a surface plasmon resonance (SPR) peak at 415 nm. TEM showed predominantly spherical nanoparticles with a main size range of 20–30 nm, a hydrodynamic diameter of 98 nm, and a zeta potential of −14.62 ± 0.04 mV. AgNPs-K showed overlapping minimum inhibitory concentration and minimum bactericidal concentration values of 1.14 µg/mL for Gram-positive species and 1.33 µg/mL for Gram-negative species. Time-kill assays showed rapid bactericidal activity after threshold concentrations were reached, with sustained suppression at 24 h. Biofilm formation was abolished at 40 µg/mL and strongly reduced at lower concentrations. AgNPs-K were non-toxic up to 400 µg/mL and improved survival in six of seven infection models. Fermented rooibos kombucha functions as an effective biosynthetic matrix for the green production of phytochemical-capped AgNPs. The resulting nanoparticles combine low-dose antibacterial and antibiofilm activity with favorable in vivo tolerability and efficacy, supporting fermentation-enabled nanobiotechnology strategies against biofilm-associated infection.