The fatality rate of invasive aspergillosis (IA) is still very high, especially in prolonged and untreated pulmonary cases. Aspergillus fumigatus is the main causative agent of IA and investigation of its metabolites could provide valuable insight into virulence factor(s) associated with this organism. We evaluated the A. fumigatus culture filtrate (CF) products generated during short- and long-term aerated and non-aerated conditions and tested for (i) inhibition of cysteine or serine proteases and (ii) cytotoxicity. In addition, the mathematical model was determined using response surface methodology (RSM) to estimate the influence of different fermentation conditions on A. fumigatus CF characteristics, predict enzyme inhibition and make possible correlations with in vivo conditions. Biosynthesis of A. fumigatus low molecular weight proteinaceous products (from 6.4 to 15.4 kDa) was observed after 6 days of growth under aerated and alkaline conditions. Also, only these CFs showed significant reduction in cell lines survival (Caco-2 and WISH 35.6% and 54.6%, respectively). Obtained results provide solid starting point for further studies that would include: (i) detailed chemical characterization of A. fumigatus CF, (ii) activity relationships and in vivo correlation with pathogenicity of prolonged pulmonary IA and (iii) possible use of biomolecules as diagnostic or therapeutic markers.