Abstract: Houseflies, Musca domestica Linnaeus, are commonest insects of both farms and homes. This species have increasingly overcrowded human dwellings, particularly in rural areas and constitute a health hazard. These flies are not only a nuisance, but they can also transport disease-causing organisms. This may lead to the spread of diseases and also mycotoxin-producing fungi. As a result, frequent exposure to the activity of houseflies will have an impact on public health. Houseflies are dominant in exposed items such as drinking water, food and human faeces. The study was conducted to determine the potential of houseflies in enhancing fungal contamination of food commodities. A total of 183 samples of houseflies (122), maize (15), porridge (19) and water (27) samples collected from Gauteng Province of South Africa were screened for fungi contamination by serial dilution technique on agar plates following DNA sequencing. In this case, 729 fungal isolates were identified with the most commonly isolated fungi belonging to the genera of Aspergillus, Fusarium and Penicillium. On the other hand, Aspergillus flavus was by far the most common, followed by A. niger, A. parasiticus and A. fumigatus. The most common Fusarium species were F. verticillioides, F. proliferatum, F. oxysporum and F. culmorum. Among the Penicillium species, Penicillium aurantiogriseum, P. brevicompactum, P. oslonii and P. verrucosum were the most common fungi. The incidence rates of fungal contamination per total fungal count isolated in houseflies, porridge, maize and water were recorded with fungal load of 2 x 108 CFU/ml, 2x 107 CFU/g, 1x 107 CFU/g and 1 x 102 CFU/g. Aspergillus, Fusarium and Penicillium species recovered were further confirmed and quantified for the production of aflatoxins (AFB1, AFB2, AFG1 and AFG2), deoxynivalenol (DON), fumonisin B1 (FB1) ochratoxin A (OTA) and zearalenone (ZEA) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The analysis of houseflies isolates by TLC revealed that FB1 was found with the highest incidence rate of 62 (73%) while, ZEA and DON had the lowest incidence rate of 15 (18%) and 8 (9%). For Fusarium isolates (66) from maize, only 11 (17%) of the extracts were positive for FB1 from F. verticillioides. The fluorescence of AFs and OTA viewed under ultra violet light revealed that some isolates were positive showing a light blue fluorescence for AFB1 and AFB2, light green fluorescence for AFG1 and AFG2, while a blue-green fluorescence was observed for OTA. According to the results on HPLC, Aspergillus flavus and A. parasiticus were found to be the main producers of AFB1, AFB2, AFG1 and AFG2, while A. carbonarius, A. niger and A.D.Tech. (Biomedical Technology