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In this work, titania nanoparticles (TiO2NPs) and silver nanoparticles (AgNPs) were combined in situ to obtain a single TiO2–Ag nanoplatform with synergistic antibacterial effects and perspectives in biological applications. To improve colloidal stability in the aqueous environment, the bifunctional (3-mercaptopropyl)-trimethoxysilane (MPTMS) and sodium 3-mercapto-1-propanesulfonate (3MPS) were used as stabilizing agents for covalent binding to TiO2NP (formation of Ti–O–Si chemical bonds) and AgNP surfaces (through the Ag–S bond), respectively. Synthesis conditions were optimized by tuning the Ag content decorating the TiO2NPs, and the colloidal stability, size, morphology, and chemical composition were studied in both solution and solid-state by UV–visible, Fourier-transform infrared, 1H NMR, X-ray photoelectron spectroscopy, field emission scanning electron microscopy-energy-dispersive X-ray spectroscopy, transmission electron microscopy, inductively coupled plasma optical emission spectrometry, dynamic light scattering, and ζ-potential. The antibacterial activities of these nanohybrids were investigated on Gram-positive and Gram-negative bacteria, and the results showed an enhanced antibacterial effect arising from the combination of TiO2NPs and AgNPs in the nanohybrid with the lower Ag content (more than 60% in the case of S. aureus exposed to TiO2–Ag), compared to the pristine TiO2NPs and control groups. Electron paramagnetic resonance with spin trapping detected the presence of hydroxyl radicals both in the nanohybrids and in the pristine TiO2NPs, supporting the hypothesis that the antibacterial effect is related to the presence of reactive oxygen species. The results of this research contribute to the synthesis of new potent antimicrobial nanohybrids for applications in more complex biological media. [ABSTRACT FROM AUTHOR]