The diagnosis of infection in patients with suspected sepsis is frequently difficult to achieve with a reasonable degree of certainty. Currently, the diagnosis of infection still relies on a combination of systemic manifestations, manifestations of organ dysfunction, and microbiological documentation. In addition, the microbiologic confirmation of infection is obtained only after 2 to 3 days of empiric antibiotic therapy. These criteria are far from perfect being at least in part responsible for the overuse and misuse of antibiotics, in the community and in hospital, and probably the main drive for antibiotic resistance. Biomarkers have been studied and used in several clinical settings as surrogate markers of infection to improve their diagnostic accuracy as well as in the assessment of response to antibiotics and in antibiotic stewardship programs. The aim of this review is to provide a clear overview of the current evidence of usefulness of biomarkers in several clinical scenarios, namely, to diagnose infection to prescribe antibiotics, to exclude infection to withhold antibiotics, and to identify the causative pathogen to target antimicrobial treatment. In recent years, new evidence with “old” biomarkers, like C-reactive protein and procalcitonin, as well as new biomarkers and molecular tests, as breathomics or bacterial DNA identification by polymerase chain reaction, increased markedly in different areas adding useful information for clinical decision making at the bedside when adequately used. The recent evidence shows that the information given by biomarkers can support the suspicion of infection and pathogen identification but also, and not less important, can exclude its diagnosis. Although the ideal biomarker has not yet been found, there are various promising biomarkers that represent true evolutions in the diagnosis of infection in patients with suspected sepsis.