In the field of sports performance, sports medicine, and physical rehabilitation, there is a great interest in the development of protocols and reliable techniques and instruments for the evaluation of strength produced by athletes. In the last ten years, women’s football has increased its popularity and participation in numerous countries, which has contributed to players developing more professionally and requiring more specific muscle strength training to improve their performance. The aim of this study was to analyze the absolute and relative test–retest reliabilities of peak muscle strength in knee flexion (FLE) and extension (EXT) controlled using a functional electromechanical dynamometer (FEMD) in a group of seventeen professional female soccer players (age = 18.64 ± 0.62 years; weight = 54.72 ± 7.03 kg; height = 1.58 ± 0.04 m; BMI = 21.62 ± 2.70 kg/m2). Peak muscle strength was measured with knee flexion (FLE) and extension (EXT) movements at a speed of 0.4 m·s−1 unilaterally in a concentric phase (CON) and an eccentric phase (ECC). No significant mean differences were found in the test–retest analysis (p > 0.05; effect size < 0.14), and high reliability was reported for peak muscle strength assessments in both the CON (ICC) = 0.90–0.95) and the ECC (ICC = 0.85–0.97). Furthermore, stable repeatability was presented for extension in the CON (CV = 7.39–9.91%) and ECC (CV = 8.65–13.64). The main findings of this study show that peak muscle strength in knee flexion and extension in CON and ECC is a measure with acceptable absolute reliability and extremely high relative reliability using the FEMD in professional female soccer players.