Purpose <p>The aims of this study were to describe the microbiological profile and antibiotic susceptibility of acute and chronic prosthetic joint infection (PJI) after total knee arthroplasty (TKA) and to propose appropriate empirical antibiotics.</p> Methods <p>We performed a retrospective review using our institution’s database to collect data from patients with PJI who underwent reoperation following a primary TKA, between 2021 and 2024. Demographic data, microbiological results and antimicrobial susceptibility testing were analysed.</p> Results <p>Forty patients met the study criteria and were included in the study. Chronic infections (&gt; 6 weeks after implantation) were the most common, accounting for 75% of the forty cases. Regardless of classification by time to infection, gram-positive organisms were the predominant causative agents. The most frequently identified pathogens were <i>Staphylococcus aureus</i> (24.2%), <i>Staphylococcus epidermidis</i> (16.7%) and <i>Staphylococcus lugdunensis</i> (13.6%). Vancomycin proved to be the most effective antimicrobial, with a susceptibility of 100% in all cases, in both acute and chronic infections. As for chronic infections, Cotrimoxazole (Sulfamethoxazole/Trimethoprim) demonstrated low levels of resistance, with 97% susceptibility among the main pathogens involved in these cases.</p> Conclusions <p>The choice of antibiotic for empirical treatment should consider the time since prosthesis implantation, as pathogen distribution and their susceptibilities differ slightly between acute and chronic infections. For acute infections, vancomycin should be considered the first-line treatment. In chronic infections, Cotrimoxazole may serve as a potential alternative treatment, due to its low resistance profile. Nevertheless, de-escalation to targeted therapy should be implemented as soon as the final culture results become available.</p>