Infective endocarditis infects the heart's inner surface, mostly the valves. It is a lethal disease with a high rate of morbidity and fatality. It mostly caused by bacteria, but fungi can also cause it. Microbiological diagnosis relies on blood culture. Molecular and biochemical indicators add to diagnosis, particularly in culture-negative patients. This study aimed to decide the role of 16s rRNA, procalcitonin, and high sensitivity C reactive protein (hsCRP) in the diagnosis of culture negative infective endocarditis (CNIE) in Assiut university hospitals. This cross-sectional study included 60 patients who were admitted to cardiac hospitals with suspected infective endocarditis according to modified DUKE criteria. A group of 20 apparently healthy subjects served as a control group for investigation of inflammatory biomarkers. We performed blood culture, biochemical markers and molecular investigations. Of the 60 patients, there were 46 (76.7%) culture positives and 14 (23.3%) culture negatives. Staphylococcus aureus was the most prevalent pathogen in culture positive patients, followed by enterococcus and klebsiella. Linezolid and imipenem were the most sensitive antibiotics for Gram-positive and Gram-negative bacteria, respectively. Amplification of bacterial 16S rRNA gene, after DNA extraction from whole blood samples, was positive in 11/14 cases (78.5%) of culture negative patients and in 42/46 cases (91.3%) of culture positive patients. The range of procalcitonin in culture positive patients was (0.32- 89) ng/ml, significantly higher than in culture negative patients (0.02- 8.8) ng/ml, and in the control group (0.01-0.08) ng/ml. hsCRP showed the same pattern. In conclusion, our data suggest that PCR was the most accurate diagnostic tool for diagnosing CNIE, followed by procalcitonin, and hsCRP, respectively.