Abstract Small non-conjugative plasmids constitute a substantial portion of the bacterial mobile genome, driving the dissemination of beneficial genes, with their transfer primarily attributed to transformation, transduction, or co-mobilization with conjugative elements 1-3 . Here we explore an understudied plasmid spread route among bacteria, mediated by intercellular membranous nanotube conduits 4 . We reveal that, unlike traditional donor-to-recipient delivery, N anotube-dependent P lasmid ex change (NPex) operates bidirectionally, enabling both plasmid donation and, to a lesser extent, plasmid acquisition. By identifying a Bacillus subtilis natural isolate deficient in NPex, we discovered a prophage-encoded factor, YokF, that blocks plasmid transmission, chiefly acting within the donor cell to inhibit plasmid donation. YokF is a nuclease that localizes to the membrane, where it interacts with a nanotube component to selectively impede plasmid transfer through degradation. Importantly, YokF homologs from various bacterial species were found to exhibit anti-NPex activity. Given their prevalence in Gram-positive bacteria, we propose that YokF homologs represent a conserved family of NPex gatekeepers that restrict plasmid flow within bacterial communities.