Airway infection and inflammation lead to the progression of obstructive lung disease in persons with cystic fibrosis (PWCF). However, cystic fibrosis (CF) fungal communities, known drivers of CF pathophysiology, remain poorly understood due to the shortcomings of traditional fungal culture. Our objective was to apply a novel small subunit rRNA gene (SSU-rRNA) sequencing approach to characterize the lower airway mycobiome in children with and without CF. Bronchoalveolar lavage fluid (BALF) samples and relevant clinical data were collected from pediatric PWCF and disease control (DC) subjects. Total fungal load (TFL) was measured using quantitative PCR, and SSU-rRNA sequencing was used for mycobiome characterization. Results were compared across groups, and Morisita-Horn clustering was performed. 161 (84%) of the BALF samples collected had sufficient load for SSU-rRNA sequencing, with amplification being more common in PWCF. BALF from PWCF had increased TFL and increased neutrophilic inflammation compared to DC subjects. PWCF exhibited increased abundance of and , while , , and Pleosporales were prevalent in both groups. CF and DC samples showed no clear differences in clustering when compared to each other or to negative controls. SSU-rRNA sequencing was used to profile the mycobiome in pediatric PWCF and DC subjects. Notable differences were observed between the groups, including the abundance of and . Fungal DNA detected in the airway could represent a combination of pathogenic fungi and environmental exposure (e.g., dust) to fungus indicative of a common background signature. Next steps will require comparisons to airway bacterial communities.