A real-time reverse-transcriptase PCR (RT-PCR) technique was developed for the rapid and specific detection and enumeration of viable Alternaria spp. in foodstuffs. The method uses Alernaria-specific primers and probe targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene. The detection limit of the real-time RT-PCR assay to detect viable Alternaria spp. in food samples was 1 CFU/g. The estimated Alternaria counts obtained by real-time RT-PCR showed a good correlation (R2 = 0.9881, P <0.01) in the range of 1–105 CFU/mL with the Alternaria counts obtained by culture methods. The applicability of the real-time RT-PCR protocol was assessed through analysis of 110 commercial food samples, including 60 fresh fruit and vegetable samples and 50 processed foodstuffs. The assay developed provides a useful tool for early detection of low concentrations of viable Alternaria spp. in naturally contaminated food samples, and could be applied as a quality and biosecurity marker of raw materials and final products in the fruits and vegetables processing industries.