The use of metal nanoparticles is gaining popularity owing to their low cost and high efficacy. We focused on green synthesis of silver nanoparticles (AgNPs) using Tinospora cordifolia (Tc) leaf extracts. The structural characteristics of Tc nanoparticles (TcAgNPs) were determined using several advanced techniques. Pharmacological activities, including antioxidant, anti-inflammatory, and antibacterial properties, were evaluated through in vitro studies. In the results, the change in sample color from yellow to brown after adding silver nitrate revealed the synthesis of TcAgNPs, and the UV–visible spectrum confirmed their formation. X-ray diffraction studies showed the presence of reducing agents and the crystalline nature of the nanoparticles. Fourier-transform infrared spectra revealed the existence of essential secondary metabolites, which act as reducing/capping agents and stabilize the nanoparticles. The size of the TcAgNPs was small (range 36–168 nm) based on the measurement method. Their negative zeta potential (−32.3 mV) ensured their stability in water suspensions. The TcAgNPs were predominantly spherical, as evidenced from scanning electron microscopy and transmission electron microscopy. Atomic absorption spectroscopy data further revealed the conversion of silver nitrate into silver nanoparticles, and thermogravimetric analysis data showed their thermal stability. The TcAgNPs showed significant DPPH/ABTS radical scavenging ability in a concentration-dependent manner (25–100 µg/mL). Membrane lysis assays showed an effective anti-inflammatory activity of the TcAgNPs. Furthermore, the TcAgNPs showed potent antibacterial effects against multidrug-resistant bacteria (Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli, and Staphylococcus aureus). The TcAgNPs treatment also exhibited antibiofilm activity against bacterial strains, in a concentration-dependent manner. Our findings demonstrate the structural characteristics of green-synthesized TcAgNPs using advanced techniques. TcAgNPs can be developed as potential antioxidant, anti-inflammatory, and antibacterial drugs.