The traditional systems of medicine have significantly become more accepted in the developed and developing countries due to their curative property, less toxicity and minimal side effects. However, several studies have shown that they are associated with microbial contaminants. This study aimed at identifying fungi in nine polyherbal medicines used for the treatment of tuberculosis in Eastern Cape Province, South Africa. Sequences of fungi DNA that encodes internal transcribed spacer (ITS) region were retrieved from the remedies. The ITS region of the fungal rRNA operon was amplified using ITS1 and ITS4 primers. The amplicons were visualized on agarose gel electrophoresis, followed by end repair and adaptor ligation. They were further purified and quantified using Library Preparation kit NEBNext® UltraT DNA Library Prep Kit for Illumina and run on Illumina’s MiSeq platform. The study revealed that the polyherbal medicines are contaminated with fungi species. The predominant mycoflora obtained belongs to different genera or species of fungi. They include Aspergillus, Penicillium, Alternaria, Candida, Ramularia, Cladosporium and Malassezia among others. Some of these organisms are capable of causing infections in immunocompromised patients. Thus, the study identified various fungal contaminants in polyherbal remedies sold to tuberculosis patients in five communities in the Eastern Cape Province of South Africa.