This study aimed to compare the dyeing capacity of annatto dye on linen textile substrates under the influence of different mordants, such as ferrous sulphate, alum, chitosan, and poly(diallyldimethylammonium chloride) (PDDACl). Annatto dye was extracted in an alcoholic medium, and the concentrations of the dye compounds bixin and norbixin were determined using ultraviolet‐visible spectrophotometry. Dyeing experiments were conducted with 10% and 20% weight of material (s.p.m.) concentrations, both on mordant‐treated substrates and on substrates pre‐bleached only. The colour evaluation indicated better colour yields for substrates pretreated with alum and PDDACl, with colour strength (K/S) values of 41.14 and 38.74, respectively, for dyeing with 20% s.p.m. However, these substrates exhibited the highest colour deviation (∆E) values in washing and lightfastness tests. The substrate pretreated with ferrous sulphate and the substrate pre‐bleached only showed K/S values of 25.99 and 18.68, respectively, with the ferrous sulphate‐treated substrate exhibiting the lowest ∆E values in washing and lightfastness tests among all evaluated substrates. Chitosan‐cationised samples showed the lowest colour yield, with a K/S of 12.51. Regarding washing and lightfastness, the pre‐bleached only substrate and the chitosan‐pretreated substrate displayed intermediate ∆𝐸 values. The dyeing kinetics of pre‐bleached substrates exhibited pseudo‐first‐order behaviour, while for ferrous sulphate‐pretreated samples, pseudo‐second‐order behaviour was observed. Langmuir's adsorption model was suitable for pre‐bleached substrate dyeing and ferrous sulphate‐pretreated sample dyeing in adsorption isotherms. However, for mordanted samples, higher adsorption intensity was observed.