Water extracts (OLE), whey protein encapsulated extracts (OLE/WPNs), and silver nanoparticles (OLE/Ag-NPs) were prepared from olive leaves of Manzenllie and Picual varieties. These preparations were characterized, and their antioxidant and biological activities on Vero and HCT-116 colorectal cells were assessed. The mechanism of action of the preparations was studied through tumor necrosis factor-α (TNF-α) and cytochrome C oxidase (Cox1) gene expression. OLE/Ag-NPs showed smaller particle sizes (14.23–15.53 nm) than OLE/WPNs (229.83–310.67 nm) and demonstrated lower aggregation due to their high Ƹ-potential of −24.86 to −27.90 mV. None of the preparations affected the viability of Vero cells (IC50 = 192.19–421.01 μg/mL), but they showed cytotoxic effects on HCT-116 cells (IC50 = 50.76–196.54 μg/mL), particularly OLE/WPNs. Moreover, the preparations from the Picual variety (OLE, OLE/WPNs, and OLE/Ag-NPs) showed regulatory effects against colon cancer on treated HCT-116 cells by upregulating Cox1 expression and downregulating TNF-α expression. Consequently, OLE/WPNs and OLE/Ag-NPs could be promising for industrial applications with potential health benefits.