The trend towards substitution of conventional transport fuels by biofuels requires additional water. The EU aims to replace 10 percent of total transport fuels by biofuels by 2020. This study calculates the water footprint (WF) of different transport modes using bio-ethanol, biodiesel or bio-electricity and of European transport if 10 percent of transport fuels is replaced by bio-ethanol. We compare results for Europe with similar goals for other regions (Africa, Asia, Latin America, the former USSR, Australia and North America). In order to provide a context, we compare results with WFs of food and cotton. In general, it is more efficient to use bio-electricity and bio-ethanol than biodiesel. Transport per train or electric car using bio-electricity (8-19 and 11-13 litres per passenger km) is more water efficient than transport by car driven by bio-ethanol (36-212) or by airplane using bio-ethanol (65-136 litres per passenger km). For cars, we find a range of a factor of ten between the most water-efficient car using bio-ethanol and the least efficient car using biodiesel. When using bio-energy, freight can be transported in the most water-efficient way by ship or train; airplanes are the least water efficient. The European goal to have 10 percent biofuel in transport in 2020 will result in a water footprint of 62 Gm3 per year. This is equal to about 10% of the current European water footprint of food and cotton consumption. Differences in per capita energy use for transport among European countries, together with differences in production systems, result in a broad range of transport-related WFs: from 60 m3 per year per capita in Bulgaria to 500 m3 per year per capita in Finland. If the same 10% biofuel target would be applied in all other regions of the world as well, the additional water footprint of China would be equivalent to 5% of the water footprint related to food and cotton consumption, in the rest of Asia 3%, in Africa 4%, in Latin America 10%, in the former USSR 22% and both in North America and Australia 52%. The global water consumption related to biofuel-based transport in this scenario would be 9% of the current global water consumption for food and cotton. The results show that a trend towards the increased application of biofuels in transport will substantially enhance the competition for fresh water resources.