Nitric oxide (NO) is an endogenous molecule with antimicrobial activity. Silver nanoparticles (AgNPs) are also well known for its antimicrobial properties. In this work, we prepared, characterized, evaluated the cytotoxicity and the antibacterial activity of alginate nanoparticles (alginate NPs) containing S-nitroso-mercaptosuccinic acid (S-nitroso-MSA, a NO donor) and/or AgNPs. AgNPs were obtained by plant mediated synthesis using green tea (Camellia sinensis), according to the green principles. Alginate NPs were obtained by ionotropic gelation with calcium ions (Ca2+) and used to incorporate the NO donor and/or AgNPs. The obtained nanoparticles containing S-nitroso-MSA and/or AgNPs have hydrodynamic size distribution in the range of 103.3 ± 2.9-414.2 ± 22.6 nm with moderate polydispersity index and negative zeta potentials. Kinetic measurements showed a sustained NO release from alginate NPs, at the mili molar range. Both S-nitroso-MSA and AgNPs were able to diffuse from the nanoparticles following a Fickian diffusion mathematical model. The cytotoxicity of the nanoparticles was evaluated towards Vero cells, and a concentration dependent toxicity was observed. The combination of the NO donor and green tea AgNPs enhanced the toxicity to the tested cells. Finally, the antibacterial activity of the nanoparticles was demonstrated against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Streptococcus mutans UA159. Interestingly, the combination of the NO donor and AgNPs into alginate NPs had a superior antibacterial activity, compared with bacteria treated with alginate NPs containing S-nitroso-MSA or AgNPs individually. Moreover, antibacterial effects were observed at nanoparticle concentrations not toxic to Vero cell. To the best of knowledge, this is the first report to demonstrate the ability of alginate NPs releasing NO and AgNPs with potent antimicrobial activity.