Diagnosis of Propionibacterium acnes bone and joint infection is challenging due to long cultivation time of up to 14 days. We retrospectively studied whether reducing cultivation time to 7 days allows accurate diagnosis without losing sensitivity. We identified patients with at least one positive P. acnes sample between 2005 and 2015 and grouped into 'infection' and 'no infection'. An 'infection' was defined when at least two samples from the same case were positive. Clinical and microbiological data including time to positivity for different cultivation methods were recorded. We found 70 cases of proven P. acnes infection with a significant faster median time to positivity of 6 days (range 2-11 days) compared to 9 days in 47 cases with P. acnes identified as a contamination (p<0.0001). In 15 of 70 (21.4%) patients with an infection, tissue samples were positive after day 7 and in 6 patients (8.6%) after day 10 when performing a blind subculture of the thioglycolate broth. Highest sensitivity was detected for thioglycolate broth (66.3%) and best positive predictive values for anaerobic agar plates (96.5%). A prolonged transportation time from the operating theatre to the microbiological laboratory did not influence time to positivity of P. acnes growth. By reducing the cultivation time to 7 days, false negative diagnosis would increase by 21.4%, thus we recommend to cultivate biopsies in bone and joint infections to detect P. acnes for 10 days with a blind subculture at the end.