Bloodstream infections (BSIs) have gained importance due to the increase in their incidence in recent years. BSIs increase the length of hospital stay, the costs associated and the patients' morbidity and mortality rates. Early diagnosis, in addition to the identification of the microorganism and its sensitivity to antimicrobial agents, has great diagnostic and prognostic importance. Several microorganisms are isolated in the bloodstream; however, multicenter studies have found Staphylococcus aureus and coagulase-negative staphylococci (CoNS) as the major etiological agents of bloodstream infections in recent decades. The examination of blood cultures is the primary means of etiological diagnosis available in clinical practice, although the step of identifying these microorganisms by conventional methods is a lengthy process. Automation is a fast and reliable option from studies that show improved performance of automated equipment, but such equipment is still not able to accurately identify the different species of CoNS because of either the slow metabolism of sugars or the variable expressions of the phenotypic traits of some of these species. The use of molecular biology techniques for bacterial identification in such cases is a solution because the results obtained are fast, accurate and sensitive, and also because the identifications performed with DNA extracted directly from blood cultures decrease the duration of the identification process significantly. This chapter aims at discussing the prevalence of Staphylococcus aureus and CoNS in BSIs by highlighting the automation techniques and molecular biology techniques as alternatives for the fast and accurate identification of Staphylococcus spp., which allows the quick start of a specific treatment.