Anaerobic co-fermentation of straw and manure is widely used for waste treatment and biogas production. However, the differences between the straw- and slurry-associated prokaryotic communities, their dynamic changes throughout the co-fermentation process, and their correlations with bioreactor performance are not fully understood. To address these questions, we investigated the prokaryotic community compositions and the dynamics of prokaryotes attached to the straw and in the slurry during co-fermentation of wheat straw and swine manure using pyrosequencing technique. The results showed that straw- and slurry-associated prokaryotes were different in their structure and function. Straw-associated prokaryotic communities were overrepresented by the phyla Spirochaetes and Fibrobacteres, while Synergistetes and Euryarchaeota were more abundant in the slurry. The straw-associated candidate class TG3, genera Fibrobacter, Bacteroides, Acetivibrio, Clostridium III, Papillibacter, Treponema, Sedimentibacter, and Lutispora may specialize in substrate hydrolysis. Propionate was the most abundant volatile fatty acid in the slurry, and it was probably degraded through syntrophic oxidation by the genera Pelotomaculum, Methanoculleus, and Methanosaeta. The protein-fermenting bacteria Aminobacterium and Cloacibacillus were much abundant in the slurry, indicating that proteins are important substrates in the co-fermentation. This study provided a better understanding of the anaerobic co-fermentation process that is driven by spatially differentiated microbiota.