Kidney stones are a common disorder associated with significant morbidity and often requires surgical intervention. Pathogenic bacteria are found in almost 40% of stones, where they form biofilms that are protected from systemic antibiotic treatments. Stone surgeries disperse biofilms resulting in up to 30% of patients developing postoperative urinary tract infections and 15% developing sepsis. This work is based on the hypothesis that chitosan, an antimicrobial polymer, can eradicate bacterial biofilms present in the stone and potentially serve as an adjunct to irrigation during stone surgery. First, fresh patient-derived kidney stone fragments (n = 56) are collected from stone surgeries. A total of 32% of stones are colonized, predominantly with Enterococcus faecalis, Escherichia coli, and Proteus mirabilis. A short, clinically relevant, chitosan treatment reduces the bacterial burden on colonized stones by over 90% in all specimens tested, regardless of stone composition and bacterial strain. To assess this approach toxicity, ex vivo human ureters and in vivo porcine bladders are exposed to topical chitosan irrigation. No toxic or pathological abnormalities other than urothelial exfoliation are noted. In conclusion, chitosan effectively disrupts kidney stone-associated bacterial biofilms with minimal urothelial toxicity and may provide an effective and safe approach to reducing postoperative complications.