To assess the need for prolonged incubation of blood culture bottles beyond five days for the diagnosis of infectious endocarditis (IE), we conducted a retrospective cohort study of 6109 sets of two blood culture bottles involving 1211 patients admitted to the Henri Mondor University Hospital for suspicion of IE between 1 January 2016 and 31 December 2019. Among the 322 patients with IE, 194 had positive blood cultures in our centre. Only one patient with a time-to-positivity blood culture of more than 120 h (5 days) was found. The main cause for the 22 patients with positive blood cultures after five days was contamination with Cutibacterium acnes. Our results do not support extending the duration of incubation of blood culture bottles beyond five days for the diagnosis of infectious endocarditis, with the exception of patients with risk factors for C. acnes infection.