Research on female offenders’ emotional sphere remains limited, with fewer studies being conducted compared to male offenders. However, investigations have identified psychobiological mechanisms in non-forensic women underlying emotional processing. One such mechanism is the activity of the autonomic nervous system. Increased parasympathetic activity has been associated with higher emotional self-regulation, social engagement, and compassion. Therefore, the present study examined heart rate (HR), vagally mediated heart rate variability (vmHRV), mood correlations, and prosocial behavior during a compassion-eliciting task using a virtual reality paradigm in female offenders (n = 31) compared to non-forensic individuals (n = 35). The primary findings showed that offenders exhibited reduced vmHRV when engaging with others’ suffering. Additionally, during the period of compassionate motivation (prosocial task), offenders revealed lower HR levels. In contrast, non-forensic individuals showed the opposite pattern. However, there were no significant mood differences observed. Finally, lower vmHRV was associated with higher donation rates only within the non-forensic group. The results suggest that offenders may encounter challenges experiencing compassion, particularly when connecting with others’ suffering and when facing compassionate motivation, as reflected by diminished parasympathetic flexibility. Furthermore, this study highlights the potential for using biomarkers when examining criminogenic needs, offering a multidisciplinary understanding of socio-affective functions, paving the way for more effective interventions.