Bloodstream infections (BSIs) are a prominent clinical problem in patients undergoing hemodialysis. These infections appear to be more common among patients who have a central line as their dialysis access and can be associated with substantial morbidity and mortality. Accurately diagnosing BSIs clearly influences patient management, but is also an important part of an infection prevention program; particularly as facility BSI rates are becoming a recognized quality measure for which dialysis facilities might be held accountable. Blood cultures remain the gold standard for diagnosing BSIs and a number of practices can affect the sensitivity and specificity of this important laboratory test. Optimizing the collection of blood cultures can assist providers with interpretation of positive blood cultures and can help minimize the impact of false-positive and false-negative cultures. This review will describe differences between BSI definitions, examine the use of blood cultures to identify these infections including the use of recommended best practices to maximize culture yield, and highlight characteristics that can assist in the clinical interpretation of positive blood cultures.