Background <p>Fungal infections are increasingly recognized as a global health concern, contributing to considerable morbidity and mortality in hospital settings. This underscores the urgent need for infection prevention and control in healthcare facilities to protect vulnerable patients from the risk of acquiring invasive fungal diseases (IFDs). Given the critical role of transmission-based precautions in limiting the spread of filamentous fungi responsible for IFDs, this study was conducted to explore the potential role of the hospital environment in the dissemination of these infections.</p> Methods <p>A total of 83 samples were collected from the air and surface of exhaust vents in the intensive care units (ICUs) of hospitals in Isfahan, Iran, to assess the presence and diversity of fungal species. Susceptibility testing against antifungal agents, including commonly used drugs and disinfectants, was performed on the identified fungal isolates. Furthermore, the antifungal resistance profiles of isolates from clinical IFD cases were compared with those of environmental isolates.</p> Results <p>Fungi were detected in 45% of air samples and 100% of exhaust vent samples, with <i>Aspergillus</i> species being the most commonly identified genus. Mucorales were also found in 17% of exhaust vent samples. <i>Aspergillus</i> spp. and <i>Rhizopus</i> spp. showed the highest resistance to Amphotericin B, and a considerable proportion of these isolates exhibited simultaneous resistance to disinfectants. A similar antifungal resistance profile was noted between <i>A. flavus</i> and some <i>R. arrhizus</i> isolates from both environmental and clinical samples.</p> Conclusions <p>The findings of this study indicate that the hospital environment, particularly exhaust vents, may act as a significant reservoir for causative agents of IFDs. This highlights the importance of environmental surveillance in preventing and controlling nosocomial fungal infections.</p>