One goal of sport psychology research is to assist athletes in adapting their training and competitive behavior to maintain or enhance high-level performance under stressful and competitive conditions. To extend this research into esports, a comprehensive understanding of the stress-performance relationship in esports is essential. This knowledge can subsequently guide future intervention studies focused on enhancing players’ performance. This study aimed to explore the relationship between playing esports in competitive settings and psychological and physiological stress among professional League of Legends players. Unlike previous research focusing on non-competitive gameplay, we examined seven male professionals during training and competition, measuring perceived motivation and match importance, as well as perceived stress, affect, emotion, heart rate, heart rate variability, and respiratory rate. Data were collected before, during, and after gameplay, evaluating in-game performance (i.e., kill-death-assist ratio, gold, and self-rated performance). Significant differences were observed, with motivation and perceived match importance being greater during competition compared to training. Our findings substantiate that both psychological and physiological stress responses intensify during competitive gameplay in contrast to training. This supports existing theoretical and empirical research, emphasizing the intricate nature of the stress-performance relationship. Practical and theoretical applications are discussed.