Conscientious objection and civil disobedience are important concepts that raise fundamental questions about the relationship between individuals and political authority. One of them is the problem of the legal status of (religious or secular) conscientious objection – in legal theory, international law and in national constitutional law. A good opportunity to analyse the concept of conscientious objection is to study one possible field of its application, i.e. the legal regulation of compulsory vaccination. The Czech Republic´s Constitutional Court has formulated a special test of applicability of conscientious objection in this context. This paper focuses not only on the relevant test but also on the much broader context of conscientious objection, the conditions of its permissibility and applicability. In addition, the paper proposes some more specific conclusions and recommendations in relation to both judicial review of conscientious objection and the approach of the legislature.