The aim of this study was to establish the role of maturation on the development of physical performance in adolescent athletes and nonathletes. The total studied sample consisted of 231 participants (131 athletes: 72 boys with an average chronological age of 13.53 ± 0.7 and 59 girls with an average chronological age of 11.97 ± 0.8; 100 nonathletes: 47 boys with an average chronological age of 13.73 ± 0.47 and 53 girls with an average chronological age of 11.93 ± 0.33), distributed according to their biological maturity stage (Pre-, Mid-, and Post-Peak Height Velocity [PHV]) and to their gender. The assessment of physical performance was performed using the following tests: Countermovement jump (CMJ), countermovement jump with arm swing (CMJA), squat jump (SJ), five-jump test (5JT), 5 m sprint (5 m), 10 m sprint (10 m), 20 m sprint (20 m), T-test, Zig Zag, and Slalom. The differences in athletes according to biological maturity were identified in all variables except for 5 m (p = 0.33) and Slalom (p = 0.07), while in nonathletes the differences were found in 5JT (p = 0.01), 5 m (p = 0.02), 10 m (p = 0.01), and 20 m (p = 0.01) tests. Additionally, a significant interaction of gender and biological maturity was detected for CMJ (p = 0.03), CMJA (p = 0.01), and Zig Zag (p = 0.05) in athletes. The findings of the current study confirm the importance of maturity status in the assessment of physical performance. As a consequence, a more rational selection of talented athletes could be provided, also enabling the timely development of physical performance in nonathletes as a “window of opportunity”.