Background: Zincite (ZnO) nanoparticles are usually prepared by decomposition of zinc acetate, hot injection and heating-up method. Microbial nanoparticle production has been developed recently, since they are clean, nontoxic and cheap. Bacteria are known as environmentally friendly nanoparticle factories using cheap, renewable carbon sources. Nanometer-sized nanoparticles are talented for different applications. For example, semiconductor nanoparticles such as Sphalerite (ZnS) are used as fluorescence probes for labeling of biological tissues. These days, magnetic nanoparticles are applied routinely in magnetic resonance imaging (MRI) as contrast-enhancement agents. Also, nano ZnO is useful in petrochemical industries. Objectives: The aim of this study was to produce nanoparticles such as ZnO, ZnS, and Zn by using biofilm of a resistant bacterium (i.e. Pseudomonas stutzeri) on a piece of pure zinc. Materials and Methods: P. stutzeri strain CS-2 was isolated from enriched soil containing methyl tertiary butyl ether (MTBE). Since the bacterium has many metal resistance genes, it was a good candidate for producing nanoparticles. A piece of pure zinc metal, sized 3.1cm was put on a solid medium (nutrient agar). The biofilm was isolated and analyzed by X-Ray diffractometer (XRD). The optimum environmental condition for producing nanoparticles was investigated. Moreover, antibacterial activity as well as magnetic property of the produced nanoparticles was studied. Finally, PCR reaction for specific PST gene was done. Results: Analysis by XRD showed 78.1% ZnO (sized 24 nm), 2.5% Zn (sized 44.88 nm) and 4.8% ZnS (sized 12 nm) produced by P. stutzeri. The nanoparticles showed anti-bacterial activity against some of the Gram positive and negative bacteria. In addition, the nanoparticles showed magnetic properties. The best condition for nano zinc production was at pH 7, in the light and at 37 °C. CzcC gene with 399Kb was detected from the isolated P. stutzeri. Conclusions: Although previous studies have shown that P. stutzeri is able to produce silver nanoparticles in silver salt solution, but in this study, for the first time ZnO, ZnS, and Zn nanoparticles were detected on a biofilm of zinc metal. Isolation of these nanoparticles by this method is very cost effective, non-toxic, clean and compatible to Eco (green chemistry).