Infective endocarditis (IE) is a heart valve infection with high mortality rates. IE results from epithelial lesions, inducing sterile healing vegetations consisting of platelets, leucocytes, and fibrin that are susceptible for colonization by temporary bacteremia. Clinical testing of new treatments for IE is difficult and fast models sparse. The present study aimed at establishing an in vitro vegetation simulation IE model for fast screening of novel treatment strategies. A healing promoting platelet and leucocyte-rich fibrin patch was used to establish an IE organoid-like model by colonization with IE-associated bacterial isolates Staphylococcus aureus, Streptococcus spp (S. mitis group), and Enterococcus faecalis. The patch was subsequently exposed to tobramycin, ciprofloxacin, or penicillin. Bacterial colonization was evaluated by microscopy and quantitative bacteriology. We achieved stable bacterial colonization on the patch, comparable to clinical IE vegetations. Microscopy revealed uneven, biofilm-like colonization of the patch. The surface-associated bacteria displayed increased tolerance to antibiotics compared to planktonic bacteria. The present study succeeded in establishing an IE simulation model with the relevant pathogens S. aureus, S. mitis group, and E. faecalis. The findings indicate that the IE model mirrors the natural IE process and has the potential for fast screening of treatment candidates.