The aims of the present work were: (1) to determine both mycobiota in raw materials and finisher poultry feed, as well as the ability to produce aflatoxin B1 by A. flavus strains, and (2) to evaluate the natural co-occurrence of aflatoxins (AFs), fumonisins (FBs), gliotoxin, diacetoxyscirpenol (DAS), HT-2 toxin, and T-2 toxin in poultry feed by LC-MS/MS. Nineteen percent of raw materials and 79% of finisher poultry feed samples exceeded the maximum allowed total fungal count (1 × 10(4) CFU g(-1)) to ensure hygienic quality. Aspergillus flavus was the only species belonging to section Flavi which was isolated while Fusarium verticilliodes was the prevalent species. Forty-seven percent of A. flavus strains were aflatoxin B1 producers and the highest frequency of aflatoxigenic strains was isolated from finisher poultry feeds. Principal component analysis showed that corn grains are closely related with total fungal and Fusarium counts. This positive relationship suggests that total fungal and Fusarium spp. counts in poultry feed might come mainly from corn grains. Regarding poultry feeds, in ground finisher type, Aspergillus spp. counts increased as water activity (aw) diminished. A positive relationship among aw, total fungal and Fusarium spp. counts was observed in both ground finisher and ground starter feed. Several mycotoxins were monitored in feeds by applying the LC MS/MS technique. One hundred percent of poultry samples were contaminated with FB1, and the highest levels were detected in pelleted finisher poultry. AFB1, gliotoxin, DAS, HT-2 toxin, and T-2 toxin were not detected in any poultry feed. The scarcity of available mycotoxicological studies from Argentinean poultry feed using a multitoxin analysis technique enhances the contribution of the findings of this report.