Clinical microbiology laboratories play an essential role in establishing an etiologic diagnosis for sepsis and bloodstream infection. Diagnosis requires multiple steps, including one or more blood cultures drawn from different sites, recovery and identification of microorganisms detected in blood culture, antimicrobial susceptibility testing of clinically significant organisms, and interpretation of results in conjunction with other clinical, radiographic, and laboratory findings. This chapter provides a summary of the clinical importance of bacteremia and fungemia, the scientific and medical principles underlying current diagnostic methods, and diagnostic approaches. Determining the clinical significance can be difficult for coagulase‐negative staphylococci, as the identity of this organism group is of little or no help. Preparation for blood culture collection and transport to the laboratory includes a number of stepwise processes. Matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry is an extensively studied approach to rapid microbial identification of blood culture isolates.