Biofilms—communities of bacteria encased in a polymer-rich matrix—confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies. Display omitted •Filamentous Pf bacteriophage are produced by Pseudomonas aeruginosa•Pf phage interact with host and microbial polymers to assemble higher order structures•Pf phage increase the viscosity of polymers in cystic fibrosis airway secretions•The Pf-induced liquid crystal biofilm matrix boosts tolerance to desiccation and antibiotics Filamentous Pf bacteriophage are often highly transcribed in P. aeruginosa biofilms. Secor et al. demonstrate that filamentous Pf bacteriophage interact with host and microbial polymers to assemble higher order liquid crystal structures. The organization of the biofilm matrix into a liquid crystal enhances biofilm adhesion, desiccation survival, and antibiotic tolerance.