The purpose of this study was to prepare a temperature-sensitive gel containing silver nanoparticles and to investigate its anti-bacterial properties in vitro. The aqueous gel was prepared using Pluronic F127 (18-22%) and Pluronic F68 (3-9%) in a cold method to obtain a proper gelation temperature at 37°C. Viscoelastic properties of the system were measured by rheological measurements and the physicochemical properties were evaluated by MJ-22 Dial-reflex metaloscope and Zetasizer Nano ZS90. The in vitro antimicrobial activity was evaluated by a disk diffusion test, minimum inhibitory concentration, and minimum bactericidal concentration. A temperature-sensitive gel containing silver nanoparticles with 20 wt% F127 and 6 wt% F68 had suitable fluidity at 25°C and was semi-solid at 37°C. Silver nanoparticle size averaged 78.0 nm. The gel optimized formulation achieved a suitable viscosity. The MIC and MBC of the gel ranged from 1.0 to 2.0 mg/L against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The activity of the gel against these three species was significantly enhanced (p < 0.05) compared to 400 mg/L Asimi standard. This optimized silver nanoparticle dosage form demonstrated a high potential for further development for the clinical treatment of bacterial vaginosis.