In the current study, extracellular biosynthesis of silver nanoparticles (AgNPs) was carried out using aqueous extracts of green Calligonum comosum stem, besides Fusarium sp. Synthesized AgNPs were characterized using ultraviolet (UV)–Vis spectrophotometer, transmission electron microscopy (TEM) and zeta potential. Moreover, biosynthesized AgNPs were estimated for the scavenging ability on DPPH radical as well as tested for their antibacterial activity using well diffusion method against Gram-positive bacteria Staphylococcus aureus. On the other hand, DNA content from untreated and AgNPs treated bacterial cells was evaluated by (UV)–Vis spectrophotometer and agarose gel electrophoresis. Results revealed the formation of AgNPs, which was first detected by color change of the reaction mixture. The characteristic surface plasmon resonance absorption was detected at 450 and 410 nm for the plant and myco-synthesized AgNPs. Furthermore, TEM micrograph and zeta sizer showed formation of spherical particles with an average size of about 105.8 and 228.4 nm for plant and myco-synthesized AgNPs, respectively. Plant-synthesized AgNPs exhibited higher scavenging of DPPH radicals than that of the myco-synthesized one. For bactericidal action, plant-synthesized AgNPs showed higher inhibition zone compared with myco-synthesized one, which was negatively correlated with the nanoparticle size. Furthermore, low DNA concentration was detected for AgNPs treated bacteria, which might be a consequence of inactivation for DNA replication. Further experimental work is required to find out if there is any correlation between nanoparticles size and efficacy against bacteria.