At present, the impact of transport on the environment constitutes a serious problem. This mainly concerns energy consumption and production of greenhouse gases (GHG) that via their participation in greenhouse effect intensification contribute to global warming. Inthis paper, the calculations provided by the EN 16258: 2012 methodology will be applied to two modes of transport: railway and road transport. Subsequently, the methodology will be applied to a case study of a selected nonelectrified railway line in comparison with parallel road transport. In particular, energy consumption and production of greenhouse gases will be monitored depending on traveling the distance between selected cities in the Czech Republic.