The endocardium and cardiac valves undergo severe impact during infective endocarditis (IE), and the formation of vegetation places IE patients at a heightened risk of embolic complications and mortality. The relevant literature indicates that 50% of IE cases exhibit structurally normal cardiac valves, with no preceding history of heart valve disease. Gram-positive cocci emerge as the predominant causative microorganisms in IE, while Gram-negative spp., persisting in the endothelium, follow pathogenic pathways distinct from those of typical IE-causing agents. Employing clinical as well as advanced microbiological and molecular assays facilitated the identification of causative pathogens, and various morphological methods were applied to evaluate heart valve damage, shedding light on the role of neutrophilic leukocytes in host defense. In this research, the immunohistochemical analysis of neutrophilic leukocyte activation markers such as myeloperoxidase, neutrophil elastase, calprotectin, and histone H3, was performed. A distinct difference in the expression patterns of these markers was observed when comparing spp.-caused and non- spp.-caused IE. The markers exhibited significantly higher expression in non- spp.-caused IE compared to spp.-caused IE, and they were more prevalent in vegetation than in the valvular leaflets. Notably, the expression of these markers in all IE cases significantly differed from that in control samples. Furthermore, we advocated the use of 16S rRNA Next-Generation Sequencing on excised heart valves as an effective diagnostic tool for IE, particularly in cases where blood cultures yielded negative results. The compelling results achieved in this study regarding the enigmatic nature of spp. IE's pathophysiology contribute significantly to our understanding of the peculiarities of inflammation and immune responses.