Dyeing faults in linen fabrics are common owing to the presence of residual lignin and pectin. Their elimination is carried out, most commonly, using alkaline methods that can damage fibers. Here we report a new enzymatic pretreatment that is more selective and ecological. Accordingly, three enzymes were tested in several concentration combinations: Viscozyme L, a carbohydrate-enzyme; Pectinex Ultra S-PL, a pectolytic enzyme; and NovoSample NS51003, a laccase from Aspergillus sp. Runs were settled by a three-factor Central Composite Design. Afterwards, the samples were dyed using a direct dye (CI Direct Blue 86) to evaluate the dyeability of the treated fabrics. The lightness variation (ΔL) and color differences (ΔE) were selected as the design responses and were analyzed using the response surface methodology. The phloroglucinol assay was applied to confirm the lignin degradation. Moreover, the results showed that a one-step enzymatic treatment using the three selected enzymes lead to a higher dye uptake (ΔL = —2.20) and lower color differences (ΔE = 0.64), compared with the non-treated sample (control: ΔL = 0 and ΔE = 2.732), using a cocktail of the three enzymes in a proportion of 3.9 ml/l of Viscozyme L, 2.34 ml/l of Pectinex Ultra SPL and 4.35ml/l of NS51003 laccase. Under these conditions the linen fabrics showed an important improvement in color uniformity after dyeing with the selected direct dye.