Elderberry fruits contain valuable components that are beneficial to human health. Owing to the high content of anthocyanins, elderberry extracts can be used as natural food colorants with health-promoting properties. Moreover, the development of new natural food dyes enables the reduction in the use of synthetic ones. Anthocyanins-rich elderberry dry extracts (EDE) were prepared from the same batch of frozen fruits applying water extraction, followed by membrane separation (batch B1) or purification by column chromatography (batch B2) and then spray-dried. Subsequently, the content of anthocyanins, flavonols, and polyphenols was determined. The extract obtained with the application of column chromatography (B2) contained 33% anthocyanins, which is more than typical market standards, whereas the extract B1 contained 14% anthocyanins. The color properties of both extracts were also determined. Since water was used as an extractant, the extracts are well soluble in water and can therefore be used as a natural food colorant. The cytotoxic activity of both extracts was additionally determined using the MTT test and the tumor cells of the A-549, A-2780, MCF-7, Caco-2 line, and Peripheral blood mononuclear cells. It was revealed that both EDEs inhibit the proliferation of cancer cells, except those of the lung cancers. Extract B2 showed a much stronger cytotoxic effect. Additionally, both extracts stimulate the proliferation of peripheral blood mononuclear cells since they may have immunostimulatory properties.