This study aimed to explore the acute effects of static stretching on the musculotendinous properties of two hamstring muscles. Twelve male volunteers underwent two testing sessions. One session was dedicated to the evaluation of the semitendinosus muscle before (PRE) and after (POST) static stretching (five sets of 30-s stretching), and the other session similarly explored the long head of biceps femoris muscle. In addition to the displacement of the myotendinous junction (MTJ), passive torque and maximal voluntary isometric torque (MVIT) were evaluated. MVIT (−8.3 ± 10.2%, p = 0.0036, d = 0.497) and passive torque (−28.4 ± 16.9%, p = 0.0003, d = 1.017) were significantly decreased POST stretching. PRE stretching, MTJ displacement was significantly greater for semitendinosus muscle than biceps femoris muscle (27.0 ± 5.2 vs. 18.6 ± 3.6, p = 0.0011, d = 1.975). After the stretching procedure, greater MTJ displacement relative changes were observed for biceps femoris muscle as compared to semitendinosus muscle (22.4 ± 31.6 vs. −8.4 ± 17.9, p = 0.0167, d = 1.252). Because of the smaller MTJ displacement PRE stretching and greater alteration POST stretching in biceps femoris muscles, the present study demonstrated muscle-specific acute responses of hamstring muscles during stretching. Although stretching acutely impairs torque production, the passive torque reduction and alteration of MTJ displacement might impact hamstring injury prevention.