A pre-workout supplement’s (PWS; 200 mg caffeine, 3.3 g creatine monohydrate, 3.2 g β-alanine, 6 g citrulline malate and 5 g branched chained amino acid (BCAA) per dose) acute effects on the alactic (jumping, sprinting, agility), lactic (Running-Based Anaerobic Sprint Test, RAST) and aerobic performance (Yo-Yo Intermittent Recovery Test Level 1, Yo-Yo IRL1 VO2max) of well-trained basketball players was investigated in this double-blind placebo-controlled study. Thirty players (age 18–31 years, height 166–195 cm, weight 70.2–116.7 kg, body fat 10.6–26.4%) were allocated to pre-workout (PWS,n= 15) or placebo (PL,n= 15) groups. Half of the participants in each group performed the evaluations without PWS or PL, while the rest consumed PWS or PL 30 min before the assessments (1st trial) and vice versa (2nd trial). Significant improvements in counter-movement jump (CMJ) (PWS: 4.3 ± 2.1%; PL: 1.2 ± 1.0%), agility (PWS: −2.9 ± 1.8%; PL: 1.8 ± 1.7%), RAST average (PWS: 18.3 ± 9.1%; PL: −2.2 ± 2.0%), minimum power (PWS: 13.7 ± 8.9%; PL: −7.5 ± 5.9%), and fatigue index (PWS: −25.0 ± 0.9%; PL: −4.6 ± 0.6%) were observed in the PWS group vs. the PL group (p< 0.05). No differences were found regarding sprinting, aerobic performance, and blood lactate concentrations. Thus, although players’ alactic and lactic anaerobic performance could be improved, peak power, sprinting and aerobic performance are not.