Vitelline membrane (VM) is a multilayered structure that surrounds the egg yolk serving to separate the yolk and the white. Due to its poor solubility in aqueous-based media, VM proteins and their biological properties have not been fully defined. In the current study, VM was hydrolyzed using different enzymes under the optimum hydrolysis conditions. Antioxidant and anti-inflammatory properties were evaluated in chemical and cellular models. Flavourzyme- and trypsin-treated samples showed the highest radical scavenging and ferric ion reducing effect (31% and 20 μM of Trolox equivalents/mg, respectively). In cellular studies, all VM hydrolysates were cyto-compatible and inhibited nitric oxide production by RAW264.7 macrophage cells significantly. Lipopolysaccharide-stimulated up-regulation of pro-inflammatory cytokines in RAW264.7 cells was suppressed by flavourzyme-treated VM. These results revealed that enzymatic hydrolysis of VM is a promising approach to produce peptides with several bioactivities (free radical scavenging, metal chelation, and anti-inflammatory) as valuable ingredients for cosmeceuticals and nutraceuticals.