Microbiological diagnosis is important for dealing with surgical infections as it provides information on causative pathogens and their antimicrobial susceptibility profiles, and thus supports appropriate antibiotic prescription. The relevance of microbiological diagnosis has increased with the emergence and dissemination of antimicrobial resistance, which narrows the repertoire of effective antimicrobial agents and complicates their selection. Microbiological diagnosis is still largely based on culturing clinical specimens to isolate the pathogens and test their antimicrobial susceptibility by phenotypic antibiogram, a well-established workflow which however takes a relatively long time and may suffer from reduced sensitivity. Recently, several novel technologies for microbiological diagnosis have been introduced in clinical practice, which can considerably shorten the time to results and possibly increase the detection sensitivity for some pathogens. These technologies can rapidly detect the presence of specific pathogens from positive blood cultures, or directly from clinical specimens, and can also inform about their antimicrobial susceptibility profile within a shorter timeframe. As such, they can provide significant guidance in the selection of appropriate antimicrobial chemotherapy, especially in epidemiological settings characterized by high resistance rates.