Many adolescents in residential care have experienced traumatic events and suffer from posttraumatic stress. Prolonged activation of neurobiological stress systems as the autonomic nervous system (ANS) and the hypothalamic-pituitary-adrenal (HPA) axis can result in long-lasting maladaptive alternations. This study investigated the effectiveness of Muse, a game-based meditation intervention, on the sympathetic nervous system (SNS), parasympathetic nervous system (PNS), and cortisol basal activity and reactivity to acute stress among adolescents with posttraumatic symptoms in residential care. The intervention consisted of two gameplay sessions a week, for 6 consecutive weeks. Seventy-seven adolescents with clinical levels of posttraumatic symptoms (10–18 years old) received either Muse as an addition to treatment as usual (n = 40) or treatment as usual alone (n = 37). We expected reduced basal activity for the SNS and cortisol and increased basal activity for the PNS. As for the response to acute stress, we expected decreased PNS and increased HPA axis reactivity. The Muse group exhibited lower basal activity for the SNS and increased HPA reactivity to acute stress. There were no differences between conditions on SNS and HPA axis activity during rest and on SNS and PNS reactivity to acute stress. Game-based meditation therapy is a promising intervention for the treatment of adolescents with posttraumatic symptoms in residential care. Implications for clinical relevance and trauma-focused treatment purposes are discussed.