Since the beginning of 2020, the COVID-19 pandemic had a major impact on rail transport in the EU. The slowdown in the spread of the COVID-19 pandemic has been achieved by reducing the mobility of the population. The reduction in mobility has had an impact on passenger transport performance. The number of national rail passengers fell by as much as 90% during the first wave of the pandemic compared to the previous year. Several operators, especially new carriers, had to close down, while rail freight operators reported a dramatic drop in volumes as many sectors slowed or even stopped production as a result of the pandemic. The second wave of the autumn 2020 pandemic has forced many countries to take further restrictive measures regarding population mobility. The outbreak of the third wave of the pandemic has prevented a rapid recovery in rail transport, especially for passenger rail services. The paper focuses on the analysis of the impact of the pandemic and the measures put in place on the development of transport performance in rail transport in the Slovak Republic during the COVID-19 pandemic. The paper proposes operational and organizational measures against the spread of the COVID-19 pandemic in railway transport in the Slovak Republic.