This study investigated the use of flax and hemp fibers in denim fabrics in terms of thermo-physiological comfort and handle-related properties. Cotton/flax and cotton/hemp blended fabrics with 2/1 twill and 3/1 twill weaves were selected and evaluated compared to pure cotton fabric. Besides, the effect of rinsing was examined. The findings revealed that the use of flax and hemp fibers improved the thermo-physiological and handle-related comfort properties of denim fabrics. Higher air permeability and water absorbency along with quick-drying behavior could be achieved in flax and hemp blended fabrics compared to pure cotton fabric. Handle-related comfort properties of flax and hemp blended fabrics indicated soft touch. Although the extension and formability values of the blended fabrics were low, they were at an acceptable level and were improved after the rinsing process.