Most hospitalized patients are carriers of biomedical devices. Infections associated with these devices cause great morbidity and mortality, especially in patients in intensive care units. Numerous strategies have been designed to prevent biofilm development on biodevices. However, biofilm formation is a complex process not fully clarified. In the current study, roughness and hydrophobicity of different biomaterials was analyzed to assess their influences on the biofilm formation of four leading etiological causes of healthcare-associated infections, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and Candida albicans, using a CDC biofilm reactor. Hydrophobic materials allowed the formation of more abundant and profuse biofilms. Roughness had effect on biofilm formation, but its influence was not significant when material hydrophobicity was considered.