Abstract Background Invasive fungal infection (IFI) is a growing cause of morbidity and mortality in oncology and transplant patients. Diagnosis of IFI is often delayed due to need for invasive biopsy and low sensitivity of conventional diagnostic methods. Fungal cell-free DNA (cfDNA) detection in plasma is a novel testing modality for the noninvasive diagnosis of IFI. Methods A novel bioinformatic pipeline was created to interrogate fungal genomes and identify multicopy sequences for cfDNA polymerase chain reaction (PCR) targeting. A real-time PCR panel was developed for 12 genera and species most commonly causing IFI. Sensitivity and specificity of the fungal PCR panel were determined using plasma samples from patients with IFI and non-IFI controls. Clinical impact of the fungal PCR panel was evaluated prospectively based on the treating team’s interpretation of the results. Results Overall, the sensitivity and specificity were 56.5% (65/115; 95% confidence interval [CI], 47.4–65.2) and 99.5% (2064/2075; 95% CI, 99.0–99.7), respectively. In the subset of patients with an optimized plasma volume (2 mL), sensitivity was 69.6% (48/69; 95% CI, 57.9–79.2). Sensitivity was 91.7% (11/12; 95% CI, 62.5–100) for detection of Mucorales agents, 56.3% (9/16; 95% CI, 33.2–76.9) for Aspergillus species, and 84.6% (11/13; 95% CI, 56.5–96.9) for Candida albicans. In a prospective evaluation of 226 patients with suspected IFI, cfDNA testing was positive in 47 (20.8%) patients and resulted in a positive impact on clinical management in 20 of 47 (42.6%). Conclusions The fungal cfDNA PCR panel offers a noninvasive approach to early diagnosis of IFI, providing actionable results for personalized care.