Nanoscale zero-valent iron (NZVI) particles were investigated in inactivating gram-positive Bacillus subtilis var. niger and gram-negative Pseudomonas fluorescens bacteria, and the fungus Aspergillus versicolor. NZVI particles were synthesized using NaBH(4) and Fe(NO(3))(3).9H(2)O, and the microbial suspensions were subjected to the treatments of NZVI particle suspensions with concentrations of 0.1, 1 and 10mg/ml for 5min. Field emission scanning electron microscope (FE-SEM) was used to characterize the synthesized NZVI particles, suspensions and the surface morphologies of the treated agents. FE-SEM images showed that the NZVI particles were spherical with a fairly uniform size of about 20-30nm, and the iron precipitates FeO(OH) appeared in needle-shape aggregates. When treated directly with NZVI particles under aerobic condition, the surfaces of microbes were quickly coated with needle-shape yellow-brown iron oxides. In this study, complete inactivation was achieved both for B. subtilis var. niger and P. fluorescens when treated with 10mg/ml NZVI particles with vigorous shaking under aerobic condition. When NZVI particle concentration decreased to 1, 0.1mg/ml, there was still a complete inactivation for P. fluorescens, while for B. subtilis var. niger the inactivation decreased to 95%, 80%, respectively. However, no inactivation was observed for the fungus A. versicolor when treated the same manner. Physical coating, disruption of membrane and generation of reactive oxygen species have played major roles in the inactivation observed.