Quaternary Trimethyl Chitosan (QTMC) and QTMC-Silver Nanoparticles (QTMC-AgNPs) have been synthesized, characterized, and tested as antibacterial agents against Staphylococcus aureus, Escherichia coli, and two plant fungi (Sclerotium rolfsil and Fusarium oxysporum). The as-prepared water-soluble QTMC was in situ reacted with silver nitrate in the presence of clean compressed hydrogen gas (3 bar) as a reducing agent to produce QTMC-AgNPs. UV-vis, ATR-FTIR, HR-TEM/SEM, XPS, DLS, XRD, and TGA/DTG were employed to assess the optical response, morphology/size, surface chemistry, particle size distribution, crystal nature, and thermal stability of the synthesized QTMC-AgNPs, respectively. The as-prepared QTMC-AgNPs were quasi-spherical in shape with an average particle size of 12.5 nm, as determined by ImageJ software utilizing HR-TEM images and further validated by DLS analysis. The development of crystalline nanoparticles was confirmed by the presence of distinct and consistent lattice fringes with an approximate interplanar d-spacing of 2.04 nm in QTMC-AgNPs. The QTMC-AgNPs exhibited significant antibacterial activity with a clear zone of inhibition of 30 mm and 26 mm around the disks against E. coli and S. aureus, respectively. In addition, QTMC-AgNPs showed highly efficient antifungal activity with 100% and 76.67% growth inhibition against two plant pathogens, S. rolfsii and F. oxysporum, respectively, whereas QTMC revealed no impact. Overall, QTMC-AgNPs showed a promising therapeutic potential and,thus, can be considered for drug design rationale.