Content: Biofouling formation on submerged structures placed in the marine environment is a worldwide problem. Many of the treatments that are used to combat biofouling provoke adverse effects on marine organisms, and thus, their use is being restricted. In this context, the society is requesting the development of alternative effective and environmental friendly antifouling treatments. The development of new materials in the last years is increasing considerably. The present work shows a guide for biofouling evaluation, from the installation and sampling of the structures in two different scenarios (the Herrera dock and the ocean-meteorological station of Pasaia in the southeastern Bay of Biscay), to the complete process of analysis including microfouling and macrofouling analysis. Different case studies are illustrated in which macrofouling formation is evaluated. This guide intends to be a useful tool that enables the discrimination of the best alternative treatments against biofouling formation.