This study aims to evaluate the effect of freeze-drying and long-term storage on the biotechnological potential of section strains. Twelve selected strains were freeze-dried and aged by accelerated storage, at 37 °C in the dark, for 2 and 4 weeks. To assess possible changes as a consequence of the ageing in the freeze-drying ampoules, morphological characteristics, mycotoxins and enzymes production, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALTI-TOF MS) spectra, and M13 phage probe fingerprinting were used as part of a polyphasic approach. Phenotypical changes were observed; nevertheless, they did not substantially affect the potential biotechnological use of these strains. The activity of hydrolytic enzymes (protease, carboxymethylcellulase, xylanase, pectinase and mannanase) was maintained or increased after freeze-drying. MALDI-TOF MS data originated spectra that grouped, for the majority of samples, according to strain independently of preservation time point. M13 profiles revealed the presence of some genetic polymorphisms after preservation. However, the three studied times still clustered for more than 50% of strains. Our results show that the studied strains maintain their biotechnological potential after preservation, with minimal phenotypic alterations. These findings provide evidence that freeze-drying preservation is a suitable option to preserve biotechnologically relevant aspergilli strains from section , and one should consider that the observed effects might be species/strain-dependent.