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Chronic wounds represent one of the complications that might occur from the disruption of wound healing process. Recently, there has been a rise in interest in employing nanotechnology to develop novel strategies for accelerating wound healing. The aim of the present study was to use a green synthesis method to obtain AgNPs/NaLS systems useful for wounds management and perform an in-depth investigation of their behavior during and post-synthesis as well as of their biological properties. The colloids obtained from silver nanoparticles (AgNPs) and commercial sodium lignosulfonate (NaLS) in a single-pot aqueous procedure have been fully characterized by UV–Vis, FT-IR, DLS, TEM, XRD, and XPS to evaluate the synthesis efficiency and to provide new insights in the process of AgNPs formation and NaLS behavior in aqueous solutions. The effects of various concentrations of NaLS (0–16 mg/mL) and AgNO3 (0–20 mM) and of two different temperatures on AgNPs formation have been analyzed. Although the room temperature is feasible for AgNPs synthesis, the short mixing at 70 °C significantly increases the speed of nanoparticle formation and storage stability. In all experimental conditions AgNPs of 20–40 nm in size have been obtained. The antimicrobial activity assessed quantitatively on clinical and reference bacterial strains, both in suspension and biofilm growth state, revealed a broad antimicrobial spectrum, the most intensive inhibitory effect being noticed against Pseudomonas aeruginosa and Escherichia coli strains. The AgNP/NaLS enhanced the NO extracellular release, potentially contributing to the microbicidal and anti-adherence activity by protein oxidation. Both AgNP/NaLS and NaLS were non-hemolytic (hemolytic index