Abstract Background Filamentous fungi are frequently recovered from respiratory cultures of individuals with CF. Methods A CF cohort database was utilized to determine filamentous fungal prevalence and risk factors. Results The prevalence of filamentous fungal isolation increased from 2.0% in 1997 to 28.7% in 2007. The odds of isolating filamentous fungi during a quarter was greater in CF adults p < 0.001, during chronic oral antibiotic use p = 0.002 and increased with each 10% drop in FEV1 percent predicted p = 0.005, while inhaled corticosteroids surprisingly decreased the likelihood p = 0.012. The direction of these effects persisted after excluding individuals with ABPA. A sub-analysis determined older age p = 0.019 and use of inhaled antibiotics p = 0.011 were independent risk factors for onset of fungal colonization. Conclusions This study suggests that isolation of filamentous fungi in CF at JHH has increased and risk factors include older age, decreased lung function, and chronic oral antibiotics.