The objective of this investigation is to synthesize and investigate zero-valent iron (ZVI) nanoparticles (NPs) for bioremediation applications. The ZVI-NPs were fabricated by chemical reduction using a ferrous salt solution with poly(N-vinylpyrrolidone) (PVP), used as a stabilizer. The synthesis was conducted with and without ultrasonic treatment. The ZVI NPs were fabricated and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD) analysis, and Fourier Transform Infrared Spectroscopy (FTIR). Experimental observations demonstrate that depending on synthesis conditions and coordination of stabilizers, NPs with different morphologies are formed. Colloidal solutions of the synthesized NPs were used in antimicrobial activity tests and biofilm formation assays for nine different control microorganisms: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 15692), Enterococcus faecalis (ATCC 29122), Klebsiella pneumoniae (laboratory isolates), Proteus vulgaris (laboratory isolates), Staphylococcus aureus (ATCC 29213), Bacillus cereus (DSMZ 4312), Bacillus subtilis (ATCC 6633), and Candida albicans (ATCC 10231). All control strains did not show antibacterial effect against PVP-stabilized ZVI NPs synthesized without ultrasonic treatment. However, biofilm results show that the highest absorbance values of the micro-organisms were tested in control wells. Although B. subtilis, E. coli, and K. pneumoniae were observed during biofilm formation, B. cereus, S. aureus, and P. aeruginosa biofilm formation reduced noticeably by Fe0/PVP-US (A1) NPs. For control strains, such as E. faecalis and C. albicans, no biofilm formation was observed. For Fe0/PVP (A2) NPs, biofilm formation of B. subtilis, E. faecalis, E. coli, K. pneumoniae, P. vulgaris, and C. albicans demonstrated positive effect, and B. cereus, S. aureus, P. aeruginosa showed negative effect. A strategic utilization of nZVI-PVP nanoparticles showed a great potential for effective, efficient, and sustainable bioremediation applications.