Background: In the bilateral Nordic hamstring exercise (NHE), hamstrings operate at relatively short lengths, which may limit the efficacy of the NHE in hamstring injury prevention. Objectives: To examine knee flexion torque and biceps femoris long head (BFLH) and semitendinosus (ST) high-density electromyography (EMG) activity during the unilateral and bilateral NHE, performed with either neutral (NHE0) or 90°-flexed (NHE90) hips. Methods: In this laboratory study, exercises were performed on a novel device at the eccentric 1-repetition maximum load defined for 90° to 15° of knee range of motion. Torque and EMG signals normalized to maximal voluntary isometric activity were compared in different phases of the exercises with statistical parametric mapping. Results: The EMG levels were lower in NHE90 than in NHE0, mainly in the second half of the movement. Knee flexor eccentric torque was higher in NHE90 than in NHE0 from the beginning to 87% of the bilateral movement, and over the entire unilateral movement. In NHE0, ST activity compared to BFLH activity was higher during the initial movement phase and lower when the movement was close to knee extension. Torque and EMG activity were generally similar in the bilateral and unilateral modes. Conclusion: If performed with neutral hips, the NHE selectively activates the BFLH near full knee extension. Performing the NHE with hips flexed to 90° is preferable when higher passive torque and ST selectivity are targeted at a longer muscle length. Performing these exercises unilaterally could help train each limb separately, with similar torque and EMG output to those of the bilateral conditions.