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In this study, fourteen coffee samples were collected from local markets in Doha, Qatar according to different variables i.e. packed or unpacked, beans or powder, roasted or raw bean samples. The coffee samples were subjected to both microbiological and chromatographical analyses. The microbiological isolation was achieved using direct plating of the coffee on malt agar extract and potato dextrose agar plates. The chromatographical analytical method was conducted using ultra-high-performance liquid chromatography-fluorescence detector (UHPLC-FLD) to quantify the contamination of four classes of aflatoxins (B1, B2, G1, and G2). In the UHPLC-FLD validation process, no interfering peaks were present and the aflatoxins G1, B1, G2, and B2 were well separated. The R2 or coefficient of determination of the calibration plots for all compounds was ≥ 0.995. The limit of quantitation for all compounds was 2 μg/kg. The precision or the relative standard deviation calculated for six replicates of samples spiked at 2 μg/kg were less than 20%, and the limit of detection was 0.17 μg/kg, 0.14 μg/kg, 0.12 μg/kg and 0.09 μg/kg for Aflatoxins G1, Aflatoxins B1, Aflatoxins G2, and Aflatoxins B2, respectively. The average recovery calculated for six replicates of samples spiked at 2 μg/kg was from 74.5–80%. The results for the chromatographical part showed that there was no contamination of aflatoxin in most of the coffee samples collected. Only two samples exhibited low concentrations of aflatoxin G, which are sample A (4.27 × 10−1 μg/kg) and sample O (1.99 × 10−2 μg/kg). Direct plating showed growth of microorganisms, such as Aspergillus niger, Mucor sp., Bacillus sp., Fusarium sp., and two yeast species. Although some of the isolated species have the potential to produce mycotoxins in coffee, they might be in the spore phase in the coffee samples and did not germinate. [ABSTRACT FROM AUTHOR]