Neuromuscular warm-up has been shown to decrease the risk of anterior cruciate ligament (ACL) injury improving muscular firing patterns. All preventive training programs described in the literature have a duration of several weeks. To date, no studies have explored the immediate effect of a neuromuscular warm-up exercise on pre-activation time of the knee stabilizer muscles. Thus, this proof-of-principle study aimed at evaluating the acute effects of a neuromuscular warm-up exercises on the electromyographic activation of knee stabilizer muscles’ activation pattern. We included 11 professional football players, mean aged 23.2 ± 4.5 years, from a Southern Italy football team. All of them underwent a standard warm-up exercise protocol at the first day of the evaluation. At 1 week, they underwent a structured neuromuscular warm-up exercise protocol. We assessed as outcome measure the pre-activation time (ms) of rectus femoris (RF), vastus medialis (VM), biceps femoris (BF), and medial hamstrings (MH) upon landing. Outcomes were assessed before and after the standard warm-up and neuromuscular warm-up. Pre-activation time of RF, VM, BF and MH significantly improved only after neuromuscular warm-up (p < 0.05); moreover, there was a significant (p < 0.05) between-group difference in pre-activation time of all muscles after the neuromuscular warm-up compared with the standard warm-up. These findings suggested that physical exercise consisting of a structured injury prevention neuromuscular warm-up might have an immediate effect in improving the activation time of the knee stabilizer muscles, thus potentially reducing the risk of ACL injury.