In the present study, Alphinia officinarum rhizome extract and zinc nitrate hexahydrate were utilized to synthesize eco-friendly and cost-effective zinc oxide (ZnO) nanoparticles (NPs). Nanoparticles of Zinc oxide have attained unique research interest among various metal oxides. Morphological characterizations of the ZnO NPs were analyzed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscope (SEM) were used to characterize the ZnO NPs. FTIR spectrum revealed the presence of functional groups in ZnO NPs. XRD, scanning electron microscope, analyses confirmed the crystal structure and average size of ZnO NPs. The antimicrobial activities of ZnO NPs were tested on microorganisms, that is, P. aeruginosa ATCC 25619, MRSA ATCC 43300, and C. albicans ATCC 10231. Moreover, the antibiofilm activity of ZnO NPs was performed against P. aeruginosa ATCC 25619, MRSA ATCC 43300, and C. albicans ATCC 10231. ZnO NPs have shown effective antimicrobial and antibiofilm activities against tested microorganisms. The results elucidated that eco-friendly and cost-effectively produced ZnO NPs could be used as coating materials and in a wide range of industrial applications, such as pharmaceutical industries and cosmetics.