Background: Due to the rising prevalence of cognitive impairment in the middle-aged and elderly population, combined with consumer demand for functional foods to improve health and well-being. Objective: This study aimed to formulate a functional cone mushroom (Termitomyces fuliginosus) (FCM) protein snack bar and evaluate its amino acid profile, phytochemical contents, biological activity and impact on cognitive function. Methods: A total of 26 middle-aged male and female participants were randomized and divided into placebo, FCM1 and FCM2 groups. Continuous consumption was performed for 6 weeks. Demographic data, body composition, cognitive function and memory were evaluated at baseline and at the end of the study period (6 weeks). Results: The event-related potential (ERP) analysis results showed a significant increase in N100 and P300 amplitude at the Fz location in participants who consumed the functional cone mushroom protein snack bar at a dose of 1 g compared to the placebo group (p = 0.015). Additionally, subjects who consumed the functional cone mushroom protein snack bar at a dose of 2 g showed a significantly increased P300 amplitude and percent accuracy of numeric working memory (p = 0.048) compared to those in the placebo group (p = 0.044). The possible underlying mechanism may involve AChE and MAO suppression activity alongside antioxidant activity. Conclusions: These data suggest that FCM can improve cognitive function and memory and may be considered for use in natural supplementation products with possible health benefits.