Mycotoxins are fungal secondary metabolites, which can cause acute and chronic toxic effects in animals and humans. The present study aimed to determine multi-mycotoxin levels in Algerian workers using urine as target. A method based on a QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction procedure followed by LC-MS/MS (liquid-chromatography tandem mass spectrometry) was optimized and validated for determination of 11 mycotoxins in 96 urine samples. Different sorbents were tested to be used in the dispersive solid-phase extraction (d-SPE) cleanup step of QuEChERS. The final method was fit-for-purpose and showed good analytical performance in terms of specificity, linearity and precision. All samples contained at least 2 mycotoxins, being toxin-2 (T-2) the most common, which was found in 92.7% of the samples, followed by zearalenone (ZEN) with 90.6% of positive samples, and ochratoxin A (OTA) with 86.4%. T-2 levels ranged from 0.3 μg/L to 36.3 μg/L, while OTA ranged from 0.3 μg/L to 3.5 μg/L and ZEN ranged from 7.6 μg/L to 126.8 μg/L. This was the first mycotoxin biomonitoring study carried out in the Algerian population. The findings highlight the need of accurate data for better risk assessment and the development of better regulation to manage mycotoxin contamination in this country.