As culture-negative implant-associated infection denote a diagnostic challenge, sonicate fluid cultures of the explanted endoprosthesis and osteosynthesis components are frequently used. However, the effect of antibiotic treatment on pathogen detection by sonication fluid cultures in implant-associated infection has not been investigated. Thus, the aim of this study was to evaluate the influence of preoperative antibiotic prophylaxis (PAP) and antibiotic therapy (AT) on sonicate fluid cultures in patients with implant-associated infection. In this retrospective study three groups were compared: (i) standard PAP, (ii) AT for at least one day, and (iii) no antibiotics before surgery. For the inclusion criteria, an established diagnostic protocol for implant-associated infection was used. Sonicate fluid cultures were validated by corresponding microbiological and histopathological samples. In 90 patients with single and multiple infections, 114 pathogens were detected. The detection rate by sonicate fluid cultures in patients receiving PAP (n = 27, 29 pathogens), AT before surgery (n = 33, 48 pathogens) and no antibiotics before surgery (n = 30, 37 pathogens) were 86.2%, 81.3%, and 86.5% (p = .778), respectively. Eleven of 114 infectious agents were detected exclusively by sonicate fluid cultures, while conventional tissue culture failed in these cases. PAP and AT do not affect intraoperative cultures in implant-associated infection. It is therefore not recommended to omit antibiotic prophylaxis in patients with implant-associated infection. Algorithms including both sonicate fluid cultures and tissue samples should be used for appropriate microbiological diagnosis of implant-associated infections.