Functionalized halloysite nanotubes were prepared by surface activation of halloysite (Hal) with sodium hydroxide and deposition of zinc oxide nanoparticles (ZnONP). The surface charge of Hal was changed from 0.18 ± 0.6 mV to −35.2 ± 2.8 mV after alkali treatment. The functionalized Hal (AT-Hal/ZnONP) was incorporated into alginate biopolymer as a reinforcing filler with different concentration of AT-Hal/ZnONP (1, 3, 5, and 7 wt% of alginate). Alginate films with AT-Hal/ZnONP exhibited a significant increase in the mechanical, water vapor barrier, and UV light barrier properties. The thermal stability of composite films has not changed after AT-Hal/ZnONP incorporation. The AT-Hal/ZnONP incorporated alginate films demonstrated strong antibacterial activity against food-borne pathogenic bacteria, Escherichia coli and Listeria monocytogenes. The nanocomposite film with 7 wt% of AT-Hal/ZnONP exhibited complete sterilization of E. coli and L. monocytogenes after 3 and 9 h of treatment, respectively.