Background: Infection is a common complication of hemodialytic treatment. Many of these infections are due to sepsis, primarily arising from the vascular access site. Simkania negevensis (S. negevensis) is an obligate intracellular bacterium belonging to the family Simkaniaceae in the Chlamydial order, which is able to survive and grow as an amoeba resisting microorganism in trophozoites and cysts of Acanthamoeba and other free-living protozoa, which probably represent its natural reservoirs. Objective: The study aimed to early diagnose and minimize the risks of Simkania negevensis infection in hemodialysis patients. Patients and Methods: This cross-sectional study was performed on 78 hemodialysis patients, and they were treated with regular hemodialysis. Patients were subjected to the following: complete history taking, full clinical assessment, laboratory investigation (complete blood count, measurement of serum IgG antibody against S. negevensisin hemodialysis patients and water samples were collected from hemodialysis circuits for detection of the occurrence of S. negevensis infection). Results:  There was no significant difference in the age and gender between the patients undergoing hemodialysis with negative water samples for Simkania compared to hemodialysis with positive water for S. negevensis. Regarding hypertension (HTN) and diabetes mellitus (DM), there was no significant difference between the patients undergoing hemodialysis with negative water samples for Simkania compared to hemodialysis with positive water for S. negevensis. IgG levels are significantly higher in patients during hemodialysis when the water samples were positive for Simkania. Conclusion: Our study detected the occurrence of S. negevensis in hemodialysis patients. When the patients were undergoing hemodialysis with positive water for Simkania, we detected significant rise of IgG antibodies against S. negevensis. Significant area under curve with significant cutoff >6.1 for IgG with sensitivity 73.3% and specificity 80.0%.