Heat Resistant Fungi (HRF) and toxigenic fungi are considered as a serious problem both in the agricultural field and for human health, due to ascospores and mycotoxins production, which can contaminate fruit and, as a consequence, adversely affect the agricultural food chain. One strategy to identify these fungi is the use of modern molecular method, which include the analysis of DNA target regions for differentiation of the fungal species. However, previously developed methods included only the identification of pure strains but not the detection of Neosartorya fischeri in artificially contaminated food samples, such as fruit or juices. Therefore, the aim of presented study was to develop a detection method of Neosartorya fischeri in contaminated strawberry fruit and juice. The other strategy is the use of phenotypic assays to determine the metabolic profile of the fungi in order to facilitate a qualitative and quantitative detection of microorganisms based on specific substrates utilization and mycotoxins production. Accordingly, the study included an evaluation of the differences in phenotype profile between N. fischeri and the phylogenetically close fungus Aspergillus fumigatus, as a strategy of their differentiation and identification. PCR detection assay was developed that revealed the presence of N. fischeri DNA in all tested contaminated samples of fruit and juice. Therefore, it can be concluded that this rapid molecular method is an important tool for the evaluation of the postharvest quality of agricultural raw materials. Moreover, the results suggest that specific metabolic and mycotoxin patterns may be used as N. fischeri detection markers and strategy in discrimination of this fungus from A. fumigatus. The results indicated that N. fischeri and A. fumigatus had a different time period of carbon sources utilization, and particularly N. fischeri presented a more efficient carbon metabolism. Mycotoxins, verruculogen and fumitremorgin C, were detected after 4 days incubation of N. fischeri. Although metabolic assays are not such fast as molecular detection approach, they allow to deeper insight into the pathways activated by heat-resistant and toxigenic fungi. Therefore, both molecular and metabolic strategies of heat-resistant fungus detection and identification are complementary and can be used to measure postharvest quality of fruit and their products.