Advances in the research of nanoparticles (NPs) with controlled charge and size are driven by their potential application in the development of novel technologies and innovative therapeutics. This work reports the synthesis, characterization, and comprehensive biological evaluation of AgNPs functionalized by ,,-trimethyl-(11-mercaptoundecyl) ammonium chloride (TMA) and trisodium citrate (TSC). The prepared AgNPs were well characterized in terms of their morphological, spectroscopic and functional properties and biological activities. The implementation of several complementary techniques allowed not only the estimation of the average particle size (from 3 to 40 nm depending on the synthesis procedure used) but also the confirmation of the crystalline nature of the NPs and their round shape. To prove the usefulness of these materials in biological systems, cellular uptake and cytotoxicity in microbial and mammalian cells were determined. Positively charged 10 nm Ag@TMA2 revealed antimicrobial activity against Gram-negative bacteria with a minimum inhibitory concentration (MIC) value of 0.17 μg/mL and complete eradication of (7 logs) for Ag@TMA2 at a concentration of 0.50 μg/mL, whereas negatively charged 10 nm Ag@TSC1 was effective against Gram-positive bacteria (MIC = 0.05 μg/mL), leading to inactivation of at relatively low concentrations. In addition, the largest 40 nm Ag@TSC2 was shown to exhibit pronounced anticancer activity against murine colon carcinoma (CT26) and murine mammary gland carcinoma (4T1) cells cultured as 2D and 3D tumor models and reduced toxicity against human HaCaT keratinocytes. Among the possible mechanisms of AgNPs are their ability to generate reactive oxygen species, which was further evaluated and correlates well with cellular accumulation and overall activity of AgNPs. Furthermore, we confirmed the anticancer efficacy of the most potent Ag@TSC2 in hiPSC-derived colonic organoids and demonstrated that the NPs are biocompatible and applicable . A pilot study in BALB/c mice evidenced that the treatment with Ag@TSC2 resulted in temporary (>60 days) remission of CT26 tumors.