Aflatoxin B1 (AFB) is a well-known carcinogen and reducing its bioavailability is of great interest for human and animal health. Several probiotic bacteria are able to bind AFB1 in vitro, including Lactobacillus rhamnosus LC-705 and Propionibacterium freudenreichii subsp. shermanii JS. A mixture of these two probiotics is used by the food and feed industry as biopreservative (Bioprofit), making it a promising candidate for future applications. Consequently, this study aims to investigate the in vitro and ex vivo ability of this probiotic mixture to bind AFB1. For in vitro experiments, probiotic mixture was suspended in an AFB1 solution (5 microM), incubated for 1 to 30 min, centrifuged, and AFB1 residues were quantitated in supernatant and pellet. For ex vivo experiments, duodenal loops of chicks were ligated and injected with either AFB1 solution alone or probiotic mixture suspension and AFB1 solution. Lumen content was centrifuged and AFB1 was quantitated in supernatant and pellet. Additionally, AFB1 was extracted from duodenal tissue to calculate tissue uptake. In vitro, 57 to 66% of AFB1 was removed from the solution by the probiotic mixture, but only 38 to 47% could be extracted from the bacterial surface. In ex vivo experiments, only up to 25% of AFB1 was bound by bacteria, and tissue uptake of AFB1 was significantly reduced when probiotic bacteria were present in the duodenal loop. Furthermore, the effect of intestinal mucus on the bacterial binding ability was investigated in vitro and was found to significantly reduce AFB1 binding by the probiotic mixture. However, probiotic mixture could only retard but not prevent AFB1 absorption in duodenal loops. Further work needs to assess the potential of probiotics in different experimental setups.