The use of nanoscale zero-valent iron (NZVI) particles in environmental remediation and wastewater treatment has recently increased. The effects of NZVI on nitrogen and phosphorus removal were examined under continuous aerobic/anaerobic conditions by employing activated sludge. NZVI did not display any measurable effect on nitrogen removal at the concentration of 50 mg/L and below. However, 200 mg/L of NZVI inhibited NH4+-N removal. The addition of NZVI at 20 mg/L and above significantly (p < 0.05) improved the phosphorous removal. The microbial activities were inhibited upon exposure to NZVI according to the ATP and reactive oxygen species (ROS). results. In comparison to control, the ATP content decreased by around 13%, 31% and 43% at the NZVI doses of 20, 50, and 200 mg/L, respectively, probably due to ROS production under NZVI exposure. Lactate dehydrogenase (LDH) release assay suggested that NZVI concentration of 200 mg/L cast adverse effects on microorganisms. Interestingly, lower concentrations of NZVI (20 and 50 mg/L) boosted the dehydrogenase activity; however, approximately 19% depression in dehydrogenase activity was detected at 200 mg/L. The high throughput 16S rDNA pyrosequencing results indicated that uncultured bacterial genera Sinobacteraceae, Xanthomonadaceae, Alcaligenaceae and Propionivibrio were sensitive to NZVI particles. 2013 Elsevier B.V. All rights reserved.