Tissue flossing aims to improve range of motion (ROM), reduce pain, and enhance injury prevention. However, evidence is lacking regarding its effects. Therefore, this study examined the effects of flossing on hamstring muscles function in comparison to dynamic stretching (DS). Seventeen healthy young men ([mean ± SD] age, 23.2 ± 1.1 years; height, 1.72 ± 0.08 m; body mass, 63.5 ± 9.3 kg) volunteered as subjects in this randomized crossover trial. The subjects received flossing, DS, and control interventions in random order at least 1 week apart to eliminate the influence of the previous intervention. Flossing involved passive twisting and active movement using a floss band (Sanctband COMPRE Floss Blueberry, Sanct Japan Co., Ltd.). DS was performed for 4 minutes in 30-second sets consisting of 15 repetitions of 2 seconds stretching. The following were measured before and after each intervention: straight leg raise (SLR) test, passive knee extension (KE) test, passive torque, passive stiffness, fascicle length in the biceps femoris long head as an indication of hamstring muscles flexibility, and maximal isometric knee flexion contraction, maximal eccentric knee extension/flexion contraction, rate of force development, and muscle activity. Flossing yielded significant improvements in the SLR test (mean difference in post-intervention changes between interventions: 5.4°, percentage change from pre- to post-value: 13.4%, p = 0.004), passive KE test (6.2°, 4.5%, p < 0.001), passive torque at end-ROM (3.8 Nm, 4.7%, p = 0.03), and maximal eccentric knee flexion contraction (14.9% body weight, 8.2%, p = 0.03) than control. Moreover, flossing yielded 2.1-fold greater improvements in the passive KE test (3.8°, 4.5%, p = 0.03) and yielded significant improvements in the maximal eccentric knee extension contraction (29.9% body weight, 13.8%, p = 0.02) than DS. Therefore, flossing on hamstring muscles is more beneficial than DS with respect to increasing ROM and muscle exertion.