In the present study, four natural textiles (cotton, linen, wool, and silk) were dyed with 14 naturally extracted dyes, and pancreatin enzyme was used in the dyeing process. The effects of pancreatin enzyme and its buffer on naturally dyed textile samples were evaluated. Two concentrations of pancreatin enzyme and buffer were used as pretreatments for dyed textiles. Proteinic fabrics showed the highest relative color strength (RCS) values of 137.23% and 132.2% when the pancreatin enzyme was applied on wool and silk dyed with pomegranate skin and bloodroot at concentrations A and B, respectively. Linen fiber dyed with catechu tree showed the highest total color difference (TCD) values with buffer (6.83) and pancreatin enzyme A (5.7) and B (6.3). This shows that there were no side effects of the pancreatin enzyme on the studied dyed textiles. By high-performance liquid chromatography (HPLC) analysis, the root extract from madder showed the presence of salicylic acid (1758.91 mg/kg extract), quercetin (844.23 mg/kg extract), ellagic acid (784.86 mg/kg extract) and benzoic acid (582.68 mg/kg extract) as main compounds. In cochineal extract the main compounds were rutin (37.732 mg/kg extract), kampherol (1915.98 mg/kg extract), myricetin (809.97 mg/kg extract), quercetin (496.76 mg/kg extract) and salicylic acid (193.87 mg/kg extract).