Background Chronic hemodialysis patients are at considerably high risk for vascular access-related blood stream infections that may be associated with serious complications. Infections are more frequent with central venous catheters and polytetrafluoroethylene grafts than with arteriovenous fistula. Aim We aim to evaluate the prevalence of vascular access infection among hemodialysis patients, risk factors, common causative microorganisms and their antibiotics sensitivity. Patients and method This cross-sectional study included 720 end-stage renal disease patients on hemodialysis recruited from six hemodialysis centers in Al-Gharbia Governorate from June 2019 to June 2020. Results The prevalence of vascular access infection was 27.77%. Sex, old age, and diabetes mellitus had a significant statistical association. Temporary catheters recorded the highest infection rate. Gram-positive bacteria were the most common organisms in 57.5% of cases, Gram negative in 39.5% while candida albicans in only 3% of the cases. The antibiotic sensitivity was higher to amikacin and imipenem. Conclusion Creation of native arteriovenous fistula is highly recommended to avoid catheter-related blood stream infections and their complications. Strict adherence to antiseptic measures and use of a rational antibiotic policy for the early diagnosis and better management of hemodialysis vascular access site infection is important.