Urinary tract infections (UTI), which are among the most frequent cases of infectious diseases, mainly affect women. The most common treatment approach involves the use of antibiotics, although this solution is not always the most suitable, mainly because of the resistance that bacterial strains develop. Proanthocyanidins are a class of polyphenols, abundantly contained in cranberry extracts, which have shown beneficial effects in the treatment of urinary tract infections, due to their anti-adhesive properties toward bacteria, with respect to the membranes of the cells of the urothelium and intestine, thus reducing their virulence. In this work, we demonstrate via microscopy and scattering measurements how a mixture of cranberry and chondroitin sulfate can form a crosslinked structure with barrier properties. By using a design of experiment (DOE), we optimized the mass ratio to obtain a precipitate between cranberry extract and chondroitin sulfate in the presence of N-acetylcysteine and hyaluronic acid. By using transepithelial electrical resistance (TEER) chambers, we confirmed the barrier properties of the best mixture obtained with the DOE. Lastly, the antibiofilm action was investigated against five strains ofEscherichia coliwith different antibiotic sensitivity. The precipitate displayed a variable inhibitory effect in biofilm formation with major effects in UTI with an antibiotic resistance profile.