Aims To obtain yeast and bacteria from ruminal fluids that possess aflatoxin B-1 (AFB(1)) detoxifying ability for use in animal feed. Methods and Results Sources of isolation were ruminal fluids of three nonfistulated dairy cows, fed diets containing cassava pulp, rice straw or distillery yeast sludge. The isolation was carried out to screen for the isolates that were active in both anaerobic and aerobic conditions. Three yeast and three bacteria isolates were selected. Up to 85% of AFB(1) was detoxified by yeast isolates and up to 60% AFB(1) reduction was evident by bacteria isolates. Two yeast isolates were identified as Kluyveromyces marxianus and one isolate as Pichia kudriavzevii. The three bacteria isolates were identified as Enterococcus faecium, Corynebacterium phoceense and C. vitaeruminis. All strains showed high biomass production when cultivated in medium with 80 g l(-1) glucose. Conclusions The isolated yeast and bacteria with AFB(1) detoxifying ability showed a good potential to be applied as an aflatoxin-detoxifying agent to ingredients used to feed dairy cattle. Significance and Impact of the Study The abilities of isolates to survive and be active in anaerobic and aerobic conditions rendered them to be active in cattle's rumen. Their biomass could be produced in bulk and used as feed supplement for aflatoxin detoxification in dairy cattle.