Direct current atmospheric pressure glow discharge (dc‐APGD) generated in contact with a flowing liquid anode (FLA) is used for continuous synthesis of size‐defined silver nanoparticles (AgNPs) modified by a (11‐mercaptoundecyl)‐N,N,N‐trimethylammonium chloride (TMA) alkanethiol self‐assembled monolayer. The impact of the operating parameters (concentration of Ag(I) ions, solution flow rate, discharge current) on the size of TMA‐AgNPs is examined. Design of experiments along with response surface methodology reveals that it is possible to obtain size‐defined TMA‐AgNPs, the smallest being 1.21 ± 0.80 nm. Furthermore, the reactive species involved in formation of TMA‐AgNPs are identified using optical emission spectrometry. TMA‐AgNPs are characterized by several other experimental techniques, which confirm production of approximately spherical, well‐dispersed TMA‐AgNPs. Additionally, attenuated total reflection Fourier transform infrared spectroscopy and UV/Vis absorption spectrophotometry confirmed that the surface of Ag nanostructures is covered by TMA. TMA‐AgNPs display antimicrobial activity toward multiple human pathogens (Escherichia coli, Staphylococcus aureus, and Candida albicans) with minimal bactericidal concentrations or minimal fungicidal concentrations of 3.90 ± 0.15, 7.79 ± 0.30, and 3.90 ± 0.15 mg/L, respectively.