<span class="paragraphSection"><div class="boxTitle">Abstract</div>Prokaryotic Argonaute proteins (pAgos) are emerging as versatile tools in nucleic acid processing; however, their roles in bacterial physiology remain poorly defined. Here, we demonstrate that overexpression of <span style="font-style: italic;">Thermus thermophilus</span> Argonaute (TtAgo) promotes transient bacterial filamentation in both <span style="font-style: italic;">T. thermophilus</span> and <span style="font-style: italic;">Escherichia coli</span> through disruption of cell division checkpoints. Scanning electron microscopy and nucleoid DNA staining revealed defective septum formation and aberrant nucleoid segregation in filamentous cells. By observing the effect of truncated TtAgo variants on septum formation and nucleoid segregation in <span style="font-style: italic;">E. coli</span>, we found that impairment of septum formation in the filamentous cells was independent of the DNA cleavage activity of the TtAgo protein. Further, we demonstrate that TtSSB interacts with TtAgo and recruits it to the replication fork, where it facilitates the DNA-binding activity of TtAgo. TtAgo acquires short DNA guides from broken double-stranded DNA and cleaves complementary chromosomal sequences, leading to DNA damage and filamentation. This filamentation triggers homologous recombination-mediated repair in <span style="font-style: italic;">T. thermophilus</span>, allowing cells to return to a rod-shaped state. These findings reveal a mechanism by which the SSB-TtAgo interaction can modulate the bacterial cell cycle and DNA repair pathways, highlighting its potential for synthetic biology and biotechnology applications.</span>