Abstract With the increasing global antibiotic uses in livestock husbandry, animal manures upon land application pose potential threats to the environments and soil microbiome. Nevertheless, effects of manures and antibiotic-administered manures on soil protists, an integral component of soil food web and primary regulators of bacteria, remain unknown. Here, we assessed impacts of cattle and poultry manures with or without an antibiotic tylosin on soil protists and their functional groups in a 130-day microcosm incubation. Protists were highly responsive to manure application, with a significant decline in their alpha diversity in all manure treatments. There were also significant temporal changes in the alpha diversity and composition of soil protists and their functional groups. Poultry manures had stronger negative influences on the community structure of protists compared to cattle manures, and more pronounced effects on protists were observed in tylosin-spiked manure treatments. Furthermore, many consumer, phototrophic and parasitic taxa were highly susceptible to all manure treatments at Day 50 and 130. Altogether, our findings demonstrate negative effects of animal manures and tylosin on soil protists. This study suggests that the applications of livestock manures and antibiotics may subsequently alter ecological functions of protists and their interactions with other soil microorganisms in agricultural systems.