In low-and middle-income countries, many infants and children remain unregistered in both civil registration and healthcare records, limiting their access to essential rights-based services, including healthcare. A novel biometric registration prototype, applying a non-touch platform using smart phones and tablets to capture physical characteristics of infants and children for electronic registration, was tested in rural Mozambique. This study assessed acceptability and perceived barriers and facilitators to the usability of this biometric registration prototype in Manhiça district, southern Mozambique. The study followed a qualitative design consisting of 5 semi-structured interviews with healthcare providers, 7 focus group discussions with caregivers of infants aged between 0 and 5 years old, and 2 focus group discussions with data collectors involved in the implementation of the biometric registration pilot project. Data were thematically analysed. The results of this study show that there is wide acceptability of the biometric registration prototype among healthcare providers and caregivers. Participants were aware of the benefits of the biometric registration prototype. The perceived benefits included that the biometric registration prototype would solve the inefficiency of paper-based registration, and the perception of biometric registration as "healthcare norm". Perceived potential barriers to the implementation of the biometric registration prototype included: myths and taboos, lack of information, lack of time, lack of father's consent, and potential workload among healthcare providers. In conclusion, the biometric prototype was widely accepted due to its perceived usefulness. However, there is a need to address the perceived barriers, and involvement of children's fathers and/or other relevant family members in the process of biometric registration.