Invasive fungal diseases (IFD) contribute significantly to worldwide morbidity and mortality, but their frequency is not well-described in some countries. The present work describes the frequency of IFD in a specialized laboratory in Colombia. A retrospective, descriptive study was implemented between March 2009 and December 2015. Results: 13,071 patients with clinical suspicion of IFD were referred during the study period, from which 33,516 biological samples were processed and analyzed using 14 laboratory methods. Diagnosis was confirmed in 1425 patients (11%), distributed according to the mycoses of interest analyzed here: histoplasmosis in 641/11,756 patients (6%), aspergillosis in 331/10,985 patients (3%), cryptococcosis in 239/8172 patients (3%), pneumocystosis in 111/1651 patients (7%), paracoccidioidomycosis in 60/10,178 patients (0.6%), and invasive candidiasis in 48/7525 patients (0.6%). From the first year of the study period to the last year, there was a 53% increase in the number of cases of IFD diagnosed. Our laboratory experienced a high frequency of IFD diagnosis, possibly attributable to the availability of a greater range of diagnostic tools. Frequency of IFD in this study was atypical compared with other studies, probably as a result of the single laboratory-site analysis. This demonstrates that implementing educational strategies helps to create a high index of clinical suspicion, while the availability and utilization of appropriate diagnostic assays assure greater reliability in identification of these cases.