The aim of this study was to estimate the aflatoxin M₁ (AFM₁) contamination in raw milk and processed milk (pasteurized or UHT) collected from two regions in Italy (Puglia and Basilicata) during a 12-year period: 2012–2023. A total of 1017 milk samples were analyzed first proceeding with screening analysis by enzyme-linked immunosorbent assay (ELISA), and suspected non-compliant samples (AFM₁ concentration higher than 0.042 µg/kg) were then analyzed by high performance liquid chromatographic with fluorimetric detection (HPLC/FLD) confirmation method. AFM₁ concentration ≥ 0.005 µg/kg (ELISA limit of quantitation) was detected in 553 of the 1017 milk samples (54.4%). AFM₁ levels exceeding the European Union maximum limit (ML) of 0.050 µg/kg were detected in 70 samples, 49 of which were determined as non-compliant samples (4.8%). Particularly high concentrations of AFM₁, exceeding 200 µg/kg, were found in four samples, three raw milk and one pasteurized. Regarding this risk exposure study, only the MOE values obtained under “high exposure scenario” were lower than 10,000, while those calculated from the overall mean values resulted as not of concern.