Local brew commonly known as Ajono is a widely consumed alcoholic beverage in Uganda. Handling practices along the production stages of the local brews may create a conducive environment for fungal colonization. This study aimed to assess fungal and aflatoxin contamination along the different production stages of Ajono from Soroti District in Eastern Uganda. A total of 180 samples were collected from the different stages along Ajono production. An interview guide was used to assess the processing practices while fungal contamination was assessed using standard microbiological methods. The study found that there are three main stages during Ajono production, namely; millet grain, fermented paste, and the liquid (Ajono) stages. During the paste fermentation stage, brewers used plastic drums (50%), pits (47%) or pots (3%) as fermentation vessels. Samples from plastic drum fermentation vessels had higher levels of fungal contamination than those from the pits and pots. Furthermore, several moulds genera including Aspergillus spp., Fusarium spp., Alternaria spp., Rhizopus spp., Penicillium spp., Cladosporium spp., and Acremonium spp. were identified along the production stages, with Aspergillus spp. as the most prevalent moulds at all stages of Ajono production. Two yeast genera; Saccharomyces spp. and Candida spp. were also isolated. Total Aflatoxin B and G group was detected along the production stages of Ajono with the highest prevalence (78.6%) seen in the liquid Ajono from pit fermenters and 68.8% in drum fermenters. Overall, all three stages of Ajono production; millet grain, fermented paste, and the liquid Ajono stages were highly contaminated with aflatoxigenic fungi which may cause adverse health effects under continued and sustained consumption of the brew