The aim of this study is to investigate the antibacterial capabilities of different coating durations of three nanoparticle (NP) coatings: molybdenum (Mo), tantalum (Ta), and zinc oxide (ZnO), and their effects on the surface characteristics of 316L stainless steel (SS). The coated substrates underwent characterization utilizing field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectrometry (EDX), and X-ray diffractometer (XRD) techniques. The antibacterial efficacy of NPs was evaluated using the agar diffusion method. The FE-SEM and EDX images confirmed the presence of nano-sized particles of Mo, Ta, and ZnO on the surface of the substrates with perfectly symmetrical spheres and a uniform distribution of the NPs. All groups demonstrated antibacterial activity, and the ability to inhibit the growth of Streptococcus mutans and Lactobacillus acidophilus bacteria. The ZnO group had the most potent antibacterial effect, followed by the Mo group, while the Ta group had the least effect. A direct-current (DC) plasma sputtering system was used to produce nano-coatings of high purity that were homogeneous, crack-free and showed no sign of delamination. Bacterial strains exposed to Mo, Ta, and ZnO coated surfaces exhibited a significant loss of viability in a time-dependent manner. The optimum sputtering time to ensure the best antibacterial properties and preserve the resources was 1 hour (h) for Mo, 3 h for Ta and 6 h for ZnO.