Aspergillus fumigatus, a thermotolerant fungus, is the main causative agent of invasive pulmonary aspergillosis (IPA) in immunocompromised patients that is associated with high mortality rates. Early diagnosis of IPA is crucial for mortality reduction and improved prognosis. An experimental inhalational model of IPA was developed in rats and the efficacy of three biomarkers, namely β-D-glucan (BDG), a panfungal marker, galactomannan (GM), a genus-specific marker, and A. fumigatus DNA, a species-specific marker was evaluated in serum and bronchoalveolar lavage (BAL) specimens at different time points postinfection for early diagnosis of IPA. BDG and GM were detected by using commercial Fungitell and Platelia Aspergillus EIA kits, respectively. A. fumigatus DNA was detected by developing a sensitive, single-step PCR assay. IPA was successfully developed in immunosuppressed rats and all animals until 5 days post-infection were positive for A. fumigatus by culture and KOH-calcofluor microscopy also showed A. fumigatus in 19 of 24 (79%) lung tissue samples. Fourteen of 30 (47%) and 27 of 30 (90%) serum and BAL specimens, respectively, were positive for all three biomarkers with 100% specificity (none of sera or BAL specimens of 12 control rats was positive for biomarkers). Our data show that BAL is a superior specimen than serum and combined detection of BDG, GM and A. fumigatus DNA provide a sensitive diagnosis of IPA in an experimental animal model. Moreover, combined detection of GM and DNA in BAL and detection of either GM or DNA in serum was also positive in 27 of 30 (90%) animals. For economic reasons and considering that the positive predictive value of BDG is low, the detection of GM and/or DNA in serum and BAL samples has the potential to serve as an integral component of the diagnostic-driven strategy in high-risk patients suspected for IPA.