The human gut microbiota plays an important role in the maintenance of human health. Factors able to modify its composition might predispose the host to the development of pathologies. Among the various xenobiotics introduced through the diet, Alternaria mycotoxins are speculated to represent a threat for human health. However, limited data are currently available about the bidirectional relation between gut microbiota and Alternaria mycotoxins. In the present work, we investigated the in vitro effects of different concentrations of a complex extract of Alternaria mycotoxins (CE; containing eleven mycotoxins; e.g. 0.153 µM alternariol and 2.3 µM altersetin, at the maximum CE concentration tested) on human gut bacterial strains, as well as the ability of the latter to metabolize or adsorb these compounds. Results from the minimum inhibitory concentration assay showed the scarce ability of CE to inhibit the growth of the tested strains. However, the growth kinetics of most of the strains were negatively affected by exposure to the various CE concentrations, mainly at the highest dose (50 µg/mL). The CE was also found to antagonize the formation of biofilms, already at concentrations of 0.5 µg/mL. LC–MS/MS data analysis of the mycotoxin concentrations found in bacterial pellets and supernatants after 24 h incubation showed the ability of bacterial strains to adsorb some Alternaria mycotoxins, especially the key toxins alternariol, alternariol monomethyl ether, and altersetin. The tendency of these mycotoxins to accumulate within bacterial pellets, especially in those of Gram-negative strains, was found to be directly related to their lipophilicity.