Abstract Background The Amazon River is one of the largest in the world and receives huge amounts of terrestrial organic matter (TeOM) from the surrounding rainforest. Despite this TeOM is typically recalcitrant (i.e. resistant to degradation), only a small fraction of it reaches the ocean, pointing to a substantial TeOM degradation by the river microbiome. Yet, microbial genes involved in TeOM degradation in the Amazon River were barely known. Here, we examined the Amazon River microbiome by analysing 106 metagenomes from 30 sampling points distributed along the river. Results We constructed the Amazon River basin Microbial non-redundant Gene Catalogue (AMnrGC) that includes ~ 3.7 million non-redundant genes, affiliating mostly to bacteria. We found that the Amazon River microbiome contains a substantial gene-novelty compared to other relevant known environments (rivers and rainforest soil). Genes encoding for proteins potentially involved in lignin degradation pathways were correlated to tripartite tricarboxylates transporters and hemicellulose degradation machinery, pointing to a possible priming effect. Based on this, we propose a model on how the degradation of recalcitrant TeOM could be modulated by labile compounds in the Amazon River waters. Our results also suggest changes of the microbial community and its genomic potential along the river course. Conclusions Our work contributes to expand significantly our comprehension of the world’s largest river microbiome and its potential metabolism related to TeOM degradation. Furthermore, the produced gene catalogue (AMnrGC) represents an important resource for future research in tropical rivers. Video abstract.