A significant portion of prosthetic joint infections with biofilm-embedded bacteria may not be adequately sampled by standard periprosthetic tissue collection techniques. The aim of the present study was to combine ultrasonication sampling procedures with standard in-traoperative sampling and cultural techniques. A total of 69 patients with implants to be removed were included in this study; a prosthetic joint infection was diagnosed or rejected according to a standardized clinical score. Intra-operative tissue specimens were cultured using standard techniques; implants were subjected to ultrasonication and sonicates used for inoculation of cultures and broad-range eubacterial PCR. According to the clinical score, a prosthetic joint infection was present in 14 of the 69 patients. 11/14 had positive cultures for tissue samples, 13/14 had positive cultures when considering in addition the results from sonicate cultures. Sonicate PCR was positive in 12/13, and negative in 1/13 (excluding one infection due to Candida albicans). Ultrasonication improved sensitivity of culture from 78.6% to 92.9%; sensitivity and specificity of PCR from sonicates was 92,3% and 100%, respectively. Combined with serially sampled periprosthetic tissue, ultrasonication of implants may help to increase the sensitivity of laboratory investigations based on cultural procedures. PCR analyses did not improve sensitivity although implementation of PCR may aid in improving the specificity of cultural detection.