Abstract: The rise in hospital-acquired infections and antimicrobial resistance underscores the urgent need for novel antimicrobial agents. Nanoparticles (NPs), such as Ag or CuO, have emerged as promising candidates for biomedical applications, including wound dressings and antimicrobial surfaces, to combat microbial infections. Combining these antimicrobial NPs with bioactive polymers like chitosan may yield synergic nanocomposites with multimodal mechanisms of action. This study aimed to synthesize silver-chitosan nanocomposites (nAgCSs) with varying Ag-to-chitosan mass ratios (1:0.3, 1:1 and 1:3) and evaluate their antimicrobial efficacy against clinically relevant bacteria Escherichia coli MG 1655, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 6538, and fungi Candida albicans ATCC 10231 and C. glabrata ATCC 90030. The antimicrobial activity of nAgCSs was assessed using a Spot test [1], a versatile and rapid test method to determine the minimum bactericidal (MBC) or fungicidal (MFC) concentrations of metal-based NPs under non-complexing conditions (in distilled water). Mechanistic studies included quantification of Ag-ion release and analyses of particle-cell interaction via confocal laser scanning microscopy (CLSM) and flow cytometry. THP-1 macrophages and HaCaT keratinocytes were used for in vitro safety assessments. Results demonstrated that the nAgCSs with an Ag-to-chitosan mass ratio of 1:3 (nAgCS_3) exhibited the highest antimicrobial efficacy. The 24-h MBC or MFC values for nAgCS_3 were 0.07–0.45 mg Ag/L for bacteria and 1.1–6.3 mg Ag/L for fungi. Notably, the antimicrobial efficacy could not be explained solely by the release of Ag ions. CLSM and flow cytometry revealed the adhesion of nAgCSs to bacterial and fungal cell surfaces, suggesting a particle-driven mode of action. Cytotoxicity assays confirmed that nAgCSs were non-toxic to THP-1 and HaCaT cells at 20 mg Ag/L. These findings highlight nAgCSs, particularly nAgCS_3, as promising candidates for antimicrobial applications.