Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Diverse prokaryotic Agos (pAgos) play potential functions in microbial defense. The functions and mechanisms of a group of full-length yet catalytically inactive pAgos, long-B pAgos, remain unclear. Here, we show that most long-B pAgos are functionally connected with distinct associated proteins, including nucleases, Sir2-domain-containing proteins and trans-membrane proteins, respectively. The long-B pAgo-nuclease system (BPAN) is activated by guide RNA-directed target DNA recognition and performs collateral DNA degradation in vitro. In vivo, the system mediates genomic DNA degradation after sensing invading plasmid, which kills the infected cells and results in the depletion of the invader from the cell population. Together, the BPAN system provides immunoprotection via abortive infection. Our data also suggest that the defense strategy is employed by other long-B pAgos equipped with distinct associated proteins.