Sorghum is the main staple food crop in developing countries, including Ethiopia. However, sorghum grain quantity and quality are affected by contaminating fungi both under field and post-harvest stage. The aim of the current study was to assessed fungal species and multi-mycotoxins associated with sorghum grain in post-harvest samples collected from eastern Ethiopia. Fungal genera of Aspergillus, Alternaria, Bipolaris, Fusarium, Mucor, Penicillium, and Rhizoctonia were recovered in the infected grain. A liquid chromatography-tandem mass spectrometric (LC-MS/MS) was used for quantification of multiple mycotoxins/fungal metabolites. Overall, 94 metabolites were detected and grouped into eight categories. All metabolites were detected either in one or more samples. Among major mycotoxins and derivatives, deoxynivalenol (137 μg/kg), zearalenone (121 μg/kg), ochratoxin A (115 μg/kg), and fumonisin B₁ (112 μg/kg) were detected with maximum concentrations, while aflatoxin B₁ had relatively lower concentrations (23.6 μg/kg). Different emerging mycotoxins were also detected, with tenuazonic acid (1515 μg/kg) occurring at the maximum concentration among Alternaria metabolites. Fusaric acid (2786 μg/kg) from Fusarium metabolites and kojic acid (4584 μg/kg) were detected with the maximum concentration among Fusarium and Aspergillus metabolites, respectively. Unspecific metabolites were recognized with neoechinulin A (1996 μg/kg) at the maximum concentration, followed by cyclo (L-Pro-L-Tyr) (574 μg/kg) and cyclo (L-Pro-L-Val) (410 μg/kg). Moreover, metabolites form other fungal genera and bacterial metabolites were also detected at varying levels. Apparently, the study revealed that sorghum grains collected across those districts were significantly contaminated with co-occurrences of several mycotoxins. Farmers should be the main target groups to be trained on the improved management of sorghum production.