Recently, different biomedical applications of aluminum oxide (Al2O3) and zinc oxide (ZnO) have been studied, and they have displayed good biocompatible behavior. For this reason, this study explores nanolaminates of [Al2O3/ZnO]n obtained by atomic layer deposition (ALD) on silicon (100) and 316L stainless steel substrates with different bilayer periods: n = 1, 2, 5, and 10. The intention is to correlate the structure, chemical bonds, morphology, and electrochemical properties of ZnO and Al2O3 single layers and [Al2O3/ZnO]n nanolaminates with their cytotoxic and biocompatibility behavior, to establish their viability for biomedical applications in implants based on the 316L SS substrate. These nanolaminates have been characterized by grazing incident X-ray diffraction (XRD), finding diffraction planes for wurtzite type structure from zincite. The chemical bonding and composition for both single layers were identified through X-ray photoelectron spectroscopy (XPS). The morphology and roughness were tested with atomic force microscopy (AFM), which showed a reduction in roughness and grain size with a bilayer period increase. The thickness of the samples was measured with scanning electron microscopy, and the results confirmed the value of ~210 nm for the nanolaminate samples. The electrochemical impedance spectroscopy analysis with Hank’s balanced salt solution (HBSS) evidenced an evolution of [Al2O3/ZnO]n/316L system corrosion resistance of around 95% in relation with the uncoated steel substrate as function of the increase in the bilayers number. To identify the biocompatibility behavior of these nanolaminate systems, the lactate dehydrogenase test was performed with Chinese hamster ovary (CHO) cells for a short system of life cell evaluation. This test shows the cytotoxicity of the multilayer compared to the single layers of Al2O3, ZnO, and 316L stainless steel. The lowest cytotoxicity was found in the single layers of ZnO, which leads to cell proliferation easier than Al2O3, obtaining better adhesion and anchoring to its surface.