Introduction: This study aimed to examine the relationship among adequate dose, serum concentration and clinical outcome in a non-selected group of hospitalized patients receiving antifungals. Methods: Prospective cross-sectional study performed between March 2015 and June 2015. Dosage of antifungals was considered adequate according to the IDSA guidelines, whereas trough serum concentrations (determined with HPLC) were considered adequate as follows: fluconazole > 11 µg/ml, echinocandins > 1 µg/ml, voriconazole 1–5.5 µg/ml and posaconazole > 0.7 µg/ml. Results: During the study period, 84 patients (65.4% male, 59.6 years) received antifungals for prophylaxis (40.4%), targeted (31.0%) and empirical therapy (28.6%). The most frequent drug was micafungin (28/84; 33.3%) followed by fluconazole (23/84; 27.4%), voriconazole (15/84; 17.9%), anidulafungin (8/84; 9.5%), posaconazole (7/84; 8.3%) and caspofungin (3/84; 3.6%). Considerable interindividual variability was observed for all antifungals with a large proportion of the patients (64.3%) not attaining adequate trough serum concentrations, despite receiving an adequate antifungal dose. Attaining the on-target serum antifungal level was significantly associated with a favorable clinical outcome (OR = 0.02; 95% CI 0.01–0.64; p = 0.03), whereas the administration of an adequate antifungal dosage was not. Conclusions: With the standard antifungal dosage, a considerable proportion of patients have low drug concentrations, which are associated with poor clinical outcome. © 2020, The Author(s).