Significance Pseudomonas aeruginosa bacteria form antibiotic-tolerant biofilms that pose significant challenges in clinical settings. Overcoming these challenges requires fundamental insights into how biofilms are formed, combined with innovative strategies to disrupt biofilms. Electron cryotomography in situ data presented here reveal the arrangement of the key P. aeruginosa adhesin CdrA at biofilm cell–cell junctions. Guided by our imaging data, we raised and characterized a CdrA-specific nanobody binder capable of disrupting these cell–cell junctions, thereby increasing the efficacy of antibiotic-mediated bacterial killing in biofilms. Together these data provide a pathway for developing effective alternative bacterial infection treatment strategies.