Introduction: Deep sternal wound infection is a rare but feared complication of median thoracotomies and is usually caused by microorganisms from the patient’s skin or mucous membranes, the external environment, or iatrogenic procedures. The most common involved pathogens are Staphylococcus aureus, Staphylococcus epidermidis and gram-negative bacteria. We aimed to evaluate the microbiological spectrum of deep sternal wound infections in our institution and to establish diagnostic and treatment algorithms. Methods: We retrospectively evaluated the patients with deep sternal wound infections at our institution between March 2018 and December 2021. The inclusion criteria were the presence of deep sternal wound infection and complete sternal osteomyelitis. Eighty-seven patients could be included in the study. All patients received a radical sternectomy, with complete microbiological and histopathological analysis. Results: In 20 patients (23%) the infection was caused by S. epidermidis, in 17 patients (19.54%) by S. aureus, in 3 patients (3.45%) by Enterococcus spp., in 14 patients (16.09%) by gram-negative bacteria, while in 14 patients (16.09%) no pathogen could be identified. In 19 patients (21,84%) the infection was polymicrobial. Two patients had a superimposed Candida spp. infection. Methicillin-resistant S. epidermidis was found in 25 cases (28,74%), while methicillin-resistant S. aureus was isolated in only three cases (3,45%). The average hospital stay for monomicrobial infections was 29.93 ± 13.69 days and for polymicrobial infections was 37.47 ± 19.18 (p = 0.03). Wound swabs and tissue biopsies were routinely harvested for microbiological examination. The increasing number of biopsies was associated with the isolation of a pathogen (4.24 ± 2.22 vs. 2.18 ± 1.6, p < 0,001). Likewise, the increasing number of wound swabs was also associated with the isolation of a pathogen (4.22 ± 3.34 vs. 2.40 ± 1.45, p = 0.011). The median duration of antibiotic treatment was 24.62 (4–90) days intravenous and 23.54 (4–70) days orally. The length of antibiotic treatment for monomicrobial infections was 22.68 ± 14.27 days intravenous and 44.75 ± 25.87 days in total and for polymicrobial infections was 31.65 ± 22.29 days intravenous (p = 0.05) and 61.29 ± 41.45 in total (p = 0.07). The antibiotic treatment duration in patients with methicillin-resistant Staphylococci as well as in patients who developed an infection relapse was not significantly longer. Conclusion: S. epidermidis and S. aureus remain the main pathogen in deep sternal wound infections. The number of wound swabs and tissue biopsies correlates with accurate pathogen isolation. With radical surgical treatment, the role of prolonged antibiotic treatment remains unclear and should be evaluated in future prospective randomized studies.