This study aimed to investigate the effect of an isometric (ISO) or Nordic hamstring exercise (NHE) intervention, alongside a sprint training program on hamstring strength, architecture, and sprinting performance in Australian footballers. Methods Twenty-five male athletes undertook NHE ( n = 13) or ISO ( n = 12) training across a 38-wk period (including preseason and in season). Biceps femoris long head (BFlh) architecture, ISO, and eccentric knee flexor strength were assessed at baseline, at the end of preseason (14 wk), and at the conclusion of the intervention. Sprint times and force–velocity profiles were determined at baseline and at the end of preseason. Results After the intervention, both groups had significant improvements in BFlh fascicle length (NHE: 1.16 cm, 95% CI = 0.68 to 1.63 cm, d = 1.88, P < 0.001; ISO: 0.82 cm, 95% CI = 0.57 to 1.06 cm, d = 1.70, P < 0.001), muscle thickness (NHE: 0.11 cm, 95% CI = 0.01 to 0.21 cm, d = 0.51, P = 0.032; ISO: 0.21 cm, 95% CI = 0.10 to 0.32 cm, d = 0.86, P = 0.002), and eccentric strength (NHE: 83 N, 95% CI = 53 to 114 N, d = 1.79, P < 0.001; ISO: 83 N, 95% CI = 17 to 151 N, d = 1.17, P = 0.018). Both groups also finished the intervention weaker isometrically than they started (NHE: −45 N, 95% CI = −81 to −8 N, d = −1.03, P = 0.022; ISO: −80 N, 95% CI = −104 to −56 N, d = −3.35, P < 0.001). At the end of preseason, the NHE group had improved their 5-m sprint time by 3.3% ± 2.0%), and their maximum horizontal velocity was 3% ± 2.1% greater than the ISO group who saw no changes. Conclusions Both ISO and NHE training with a periodized sprinting program can increase BFlh fascicle length, thickness, and eccentric strength in Australian footballers. NHE training also improves 5-m sprint time and maximum velocity. However, both interventions reduced ISO strength. These findings provide unique, contextually relevant insights into the adaptations possible in semiprofessional athletes.