The present study deals with the toxicity assessment of two differently synthesized zero valent iron nanoparticles (nZVI, chemical and biological) as well as Fe2+ ions on Artemia salina at three different initial concentrations of 1, 10, and 100 mg/L of these particles. The assessment was done till 96 h at time intervals of 24 h. EC50 value was calculated to evaluate the 50% mortality of Artemia salina at all exposure time durations. Between chemically and biologically synthesized nZVI nanoparticles, insignificant differences in the level of mortality were demonstrated. At even 24 h, Fe2+ ion imparted complete lethality at the highest exposure concentration (100 mg/L). To understand intracellular oxidative stress because of zero valent iron nanoparticles, ROS estimation, SOD activity, GSH activity, and catalase activity was performed which demonstrated that ionic form of iron is quite lethal at high concentrations as compared with the same concentration of nZVI exposure. Lower concentrations of nZVI were more toxic as compared with the ionic form and was in order of CS-nZVI > BS-nZVI > Fe2+. Cell membrane damage and bio-uptake of nanoparticles were also evaluated for all three concentrations of BS-nZVI, CS-nZVI, and Fe2+ using adult Artemia salina in marine water; both of which supported the observations made in toxicity assessment. This study can be further explored to exploit Artemia salina as a model organism and a biomarker in an nZVI prone aquatic system to detect toxic levels of these nanoparticles. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1617–1627, 2017. © 2017 Wiley Periodicals, Inc.