In this study, synthesis of silver nanoparticles (AgNPs) was carried out utilizing the red and green parts of the pistachio hulls then their several biological activities were investigated. The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) activities of the AgNPs synthesized from red pistachio hulls extracts (PhR-AgNPs) and green pistachio hulls extracts (PhG-AgNPs) were investigated. The DPPH scavenging capability at 200 mg/L concentration of PhR-AgNPs was around 93.01% however PhG-AgNPs displayed 91.00%. The synthesized PhR-AgNPs and PhG-AgNPs acted on the E. coli plasmid DNA, causing its complete degradation and exhibiting effective chemical nuclease activity. Furthermore, PhR-AgNPs and PhG-AgNPs showed quite good antimicrobial activity against the studied strains with a range of the minimum inhibition concentration (MIC) of 8–16 mg/L. Moreover, it was observed that both pistachio hulls coated with AgNPs were highly effective in inhibiting the biofilm generation studied strains. Moreover, PhR-AgNP and PhG-AgNP displayed a completely inhibition effect on cellular viability of E. coli with 100% at 125 mg/L.