Silver nanoparticles (Ag-NPs) have wide applicability as antimicrobial, biomedical, and anti-cancer drug delivery systems. This study aimed to synthesize Ag-NPs using green methodology to assess their antimicrobial properties and toxicity. Ag-NPs were prepared using doxycycline, an antibiotic serving as a reducing and capping agent. The synthesized Ag-NPs were characterized by ultraviolet-visible spectrophotometry, X-ray diffraction, scanning electron microscopy, and Fourier-transform infrared spectrometry. The antimicrobial efficacy of doxycycline-mediated Ag-NPs was assessed on Candida species in vitro and for toxicity in male albino mice in vivo. The Ag-NPs showed a surface plasmon resonance at 411 nm, indicating a 90 nm average size and spherical shape. Toxicity was tested on mouse organs (liver, kidney, spleen, heart and stomach) using three Ag-NP doses (50, 100, 200 mg/kg) over 14 days. The synthesized Ag-NPs produced large inhibition zones against two well-known fungal species, C. albicans and C. tropicalis, demonstrating their antimicrobial potential. The Ag-NPs showed varying degrees of toxicity in different mouse organs, depending on the administered dose, with more pronounced adverse effects observed at higher concentrations. Periodic administration of Ag-NPs at low-dose volumes holds promise as a safe approach to their use as antimicrobial agents. Low-dose Ag-NPs are minimally toxic and show strong antimicrobial efficacy.