Recently, fabricated nanoparticles (NPs), which can efficiently penetrate biological systems, have found increased usage in the health and hygiene industries. Microbial enzymes and proteins have recently shown their potential to act as reducing agents for the production of NPs, thereby providing an alternative to physical and chemical methods. Not only is this approach efficient and cost-effective, but it also produces a minimal ecological footprint. In this study, zinc oxide nanoparticles (ZnO NPs) were synthesized using probiotic bacteria (Lactobacillus fermentum) as the reducing and capping agent. Several analytical methods, including Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), ultraviolet–visible spectroscopy (UV–Vis), and atomic force microscopy (AFM), were used to analyze the produced ZnO NPs. The SEM analysis confirmed the spherical form of the nanoparticles and estimated their average size to be between 100 and 120 nm. FT-IR analysis verified that the ZnO NPs’ surfaces contained many functional groups. X-ray diffraction examination evidenced that the biogenically produced nanoparticles were crystalline. AFM analysis revealed that the nanoparticles’ size was about 90–100 nm. The maximum absorption peak, determined via a UV–visible spectrophotometer, was 510 nm. The synthesized ZnO NPs’ antimicrobial activity against various bacterial strains was tested, and the highest level of antimicrobial activity was noted against a Vibrio harveyi strain. The maximum concentration, namely, 20 mM of ZnO NPs, showed the highest antimicrobial activity. These observations indicate that the synthesized ZnO NPs possess remarkable antimicrobial potency. This method is an efficient, environmentally friendly, cost-effective approach for producing ZnO NPs that are useful for various biomedical applications.