Biocolourants have been investigated as alternatives to synthetic dyes. However, natural origin per se is not a label of harmlessness and research is needed to obtain safe dyes. We studied the cytotoxicity of the extracts from fungal (Cortinarius semisanguineus, Tapinella atrotomentosa) and plant (Tanacetum vulgare, Salix phylicifolia) sources and the woollen fabrics dyed with the extracts. Cytotoxicity in vitro using hepa-1 mouse hepatoma cells for 24 h and 72 h exposure was observed as the highest tolerated dose. All biocolourants produced intensive colour on fabrics with fastness properties from moderate to good. The Salix and Cortinarius samples did not show any cytotoxic effects, whereas the Tanacetum and Tapinella samples had slightly higher test values but were not interpreted as being significantly toxic. Higher than zero values of the undyed fabrics showed the importance of examining their toxicity as well. It was found that the cytotoxicity of the samples dyed with the biocolourants did not differ significantly from the undyed wool fabric. The concentrations of dyes used in the assays were very low, imitating the dose of the user. In addition to colouring properties, natural dyes may have pharmaceutical and antibacterial properties which would enhance the interest in using them in products for added value.