Competitive football players who undergo strenuous training and frequent competitions are more vulnerable to psychological disorders. Probiotics are capable of reducing these psychological disorders. The present study aimed to determine the effect of daily probiotics supplementation on anxiety induced physiological parameters among competitive football players. The randomized, double-blinded, placebo-controlled trial was conducted on 20 male footballers who received either probiotics (Lactobacillus Casei Shirota strain 3 × 1010 colony forming units (CFU) or a placebo drink over eight weeks. Portable biofeedback devices were used to measure the electroencephalography, heart rate, and electrodermal responses along with cognitive tests at the baseline, week 4, and week 8. Data were statistically analyzed using mixed factorial ANOVA and results revealed that there is no significant difference between the probiotic and placebo groups for heart rate (61.90 bpm ± 5.84 vs. 67.67 bpm ± 8.42, p = 0.09) and electrodermal responses (0.27 µS ± 0.19 vs. 0.41 µS ± 0.12, p = 0.07) after eight weeks. Similarly, brain waves showed no significant changes during the study period except for the theta wave and delta wave at week 4 (p < 0.05). The cognitive test reaction time (digit vigilance test) showed significant improvement in the probiotic group compared to the placebo (p < 0.05). In conclusion, these findings suggest that daily probiotics supplementation may have the potential to modulate the brain waves namely, theta (relaxation) and delta (attention) for better training, brain function, and psychological improvement to exercise. Further research is needed to elucidate the mechanism of current findings.