Background Culturing of the sonication fluid of removed implants has proven to be more sensitive than conventional periprosthetic tissue culture for the microbiological diagnosis of prosthetic joint infection. Since bacteria surviving on antibiotic-loaded cement spacers used in a two-stage exchange protocol for infected arthroplasties may cause the persistence of infection, in this study we asked whether the sonication also could be used to identify bacteria on antibiotic-loaded cement spacers removed at the second surgical stage during a two-stage exchange procedure to confirm whether or not the prosthetic joint infection had been eradicated. Methods We cultured the sonication fluid of cement spacers that had been originally implanted in a two-stage exchange protocol in 21 patients (mean age, 66 years) affected by prosthetic joint infection (16 total knee prostheses and 5 hip prostheses). The cement spacers were vortexed for 30 seconds and then subjected to sonication (frequency 35–40 KHz). The resulting sonicate fluid was cultured for aerobic and anaerobic bacteria. Results The sonication fluid culture of the removed spacer was positive in six patients (29%), with isolation of methicillin-sensible Staphylococcus Aureus (MSSA) in three cases, methicillin-resistant Staphylococcus Aureus (MRSA) in one case and Pseudomonas Aeruginosa in two cases. In three of these positive cases, the traditional culture of periprosthetic tissue was negative. Two patients with positive sonication culture of the spacer were successfully treated by early debridement of the revision prosthesis and systemic antibiotic therapy. In three patients a knee arthrodesis was planned and performed as the second surgical stage. In two of them the infection was caused by highly resistant Pseudomonas Aeruginosa. The other patient with a MSSA infection had been poorly compliant with the systemic antibiotic therapy due to her mental impairment. The patient originally affected by MRSA infection of his primary hip arthroplasty developed recurrent infection of his revision prosthesis and eventually underwent Girdlestone arthroplasty. Conclusions The sonication culture can be used to discover any bacteria on the antibiotic-loaded cement spacer during a two-stage exchange protocol, thus permitting the adoption of timely treatment options, such as the early prosthetic debridment.