Infective endocarditis (IE) is a serious infection of the inner surface of heart, resulting from minor lesions in the endocardium. The damage induces a healing reaction, which leads to recruitment of fibrin and immune cells. This sterile healing vegetation can be colonized during temporary bacteremia, inducing IE. We have previously established a novel in vitro IE model using a simulated IE vegetation (IEV) model produced from whole venous blood, on which we achieved stable bacterial colonization after 24 h. The bacteria were organized in biofilm aggregates and displayed increased tolerance toward antibiotics. In this current study, we aimed at further characterizing the time course of biofilm formation and the impact on antibiotic tolerance development. We found that a Staphylococcus aureus reference strain, as well as three clinical IE isolates formed biofilms on the IEV after 6 h. When treatment was initiated immediately after infection, the antibiotic effect was significantly higher than when treatment was started after the biofilm was allowed to mature. We could follow the biofilm development microscopically by visualizing growing bacterial aggregates on the IEV. The findings indicate that mature, antibiotic-tolerant biofilms can be formed in our model already after 6 h, accelerating the screening for optimal treatment strategies for IE.