The Republic of Croatia is a land of great biodiversity. In a traffic – geographical sense, it is a transit country crossed by many international transport corridors. Highways, fast roads and well-developed transport infrastructure are therefore important factors for economic development. Nevertheless, it is also necessary to take account of the conservation of nature. By habitat fragmentation routes (roads and railways) divide natural entities into smaller parts (fragments), thereby negatively affecting biodiversity. The aim of this paper was to identify the impacts of transport infrastructure and transport on the plant and animal world and possible measures of protection. The negative effects of fragmentation on plant life are manifested in the division of habitats into smaller parts (fragments) and the creation of the so-called edge effect. Negative effects on animals are casualties in traffic or their movement is blocked due to fences along highways and two-lane expressways. This reduces populations or even leads to extinction of some sensitive species. There are various technical measures to mitigate the negative effects of traffic and roads, namely: 1. wildlife crossings - green bridges, 2. passages for animals, 3. animal protection fences. Animal protection as well as the preservation of the integrity and connectivity of habitats are regulated by the Law on Nature Protection as the umbrella law, Bylaws and International Conventions. Despite the negative effects of fragmentation of the habitat, it is impossible to prevent the construction of transport infrastructure and other supporting structures. To date, the quality of highway construction has progressed so that the highways today are much safer and of higher quality, and besides providing quality driving, when designing and constructing, account is taken of nature protection and the application of various technical measures to mitigate the negative impacts of roads on the plant and animal world.