Fungi are responsible for approximately 10% of nosocomial infections, with Candida spp. being the most prevalent etiological agent. Immunocompromised individuals are more susceptible to fungal infections. An early determination of the etiology of the infection is extremely important for these patients because it provides valuable information for choosing the best therapeutic scheme and may increase the chances of recovery. Due to the low sensitivity of direct examination, the determination of the etiology of a fungal infection often requires culture of biological specimens for isolation and identification of the disease causative agent. Blood culture is not effective for diagnosis of invasive fungal infections, even when serial specimens are analyzed. Serological identification of fungal cell wall proteins (galactomannan, mannan, β-glucan) are considered the first line diagnosis of invasive fungal infections; however, it has low sensitivity because the antigens are rapidly removed from the bloodstream. Molecular biology diagnostic methods have been developed as an alternative for the - detection of fungal infections that are difficult or impossible to detect by conventional microbiological and serological methods. In this chapter, we provide an up-to-date review of nosocomial fungal infections, focusing on the occurrence, etiology, risk factors, clinics, as well as the conventional and molecular methods of diagnosis. We also discuss the use of molecular markers for the detection of fungal infection.