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Background: Infective endocarditis (IE) is a severe cardiac infection involving microbial colonization of endocardial surfaces, primarily valves. Diagnosis is complex, requiring identification of the primary cardiac site and assessment of systemic complications. Objectives: This study aims to identify, monitor, and characterize nosocomial pathogens causing IE to enhance therapeutic strategies. Methods: This cross-sectional study was performed on 20 patients with IE in Namazi, Shahid Faqihi, and Qalb al-Zahra hospitals in Shiraz. Participants were Iranian adults (aged 18 - 80) presenting with initial symptoms of IE according to the modified Duke criteria. The study period was 18 months. All cases were evaluated by blood culture test. Then, by biochemical methods, known microorganisms were assessed. Finally, the genome of all known bacteria in IE was amplified by polymerase chain reaction (PCR) and then sequenced. GraphPad Prism 9.0 was used for statistical analysis. The chi-square and Fisher's exact tests were used to assess correlations (P-value < 0.05 considered significant). Results: Blood culture analysis in this IE cohort revealed 85% positivity (17/20 cases). Among positive cultures, Staphylococcus aureus (25%), Streptococcus spp. (20%), and S. epidermidis (15%) were the most prevalent pathogens. Notably, staphylococci collectively accounted for 74% of all pathogenic isolates. The affected population was predominantly male (highest percentage) within the 41 -60 year age range. Furthermore, antimicrobial susceptibility testing indicated markedly elevated rates of antibiotic resistance among the identified microorganisms. Conclusions: This study identifies Staphylococcus species, particularly S. aureus, as the predominant IE pathogens. The detection of fastidious organisms in culture-negative cases highlights the need to expand the etiological spectrum considered, especially in region-specific contexts. The high prevalence of antimicrobial resistance among isolates necessitates enhanced microbial surveillance and robust antibiotic stewardship. Rapid pathogen identification and molecular characterization remain critical for optimizing IE diagnostic and therapeutic management.