Jana Mulačová studied at the Faculty of Nuclear and Physical Engineering at Czech Technical University in Prague, focusing on satellite laser ranging and atmospheric simulations. She completed the international SpaceMaster program with a double master's degree in Technical Cybernetics from CTU Prague and Space Science and Technology from Technical University of Lulea in Sweden, specializing in neural networks for spacecraft anomaly detection. Her career began programming spacecraft simulators for the European Space Control Center (ESOC) in Darmstadt, working on missions including Rosetta, Venus Express, and Mars Express. She then advanced through roles at the Danish National Space Institute (DTU Space), from software prototyping to Product Assurance and Quality Management, eventually becoming Chief Quality Officer for Denmark's part of the ITER fusion reactor project.

In 2017, she returned to ESOC as Product Assurance representative for numerous ESA missions, specializing in anomaly investigation and risk management across space operations. Beyond her technical work, Jana co-created the viral hit "European Space-ody" alongside Queen guitarist and astrophysicist Sir Brian May, which became ESA's unofficial anthem. For this contribution, she received the ESA Champion title and an international ESA award, granting her special access to key ESA information and authorization to disseminate it to member states. Since 2024, she has worked in Knowledge and Innovation Management under ESOC's Transformation Office.