Objective: Identify changes in the prevalence and antimicrobial resistance patterns of potentially pathogenic bacteria in urine cultures during a 2-year antimicrobial stewardship intervention program in nursing homes (NHs). Design: Before-and-after intervention study. Setting: The study included 27 NHs in North Carolina. Methods: We audited all urine cultures ordered before and during an antimicrobial stewardship intervention. Analyses compared culture rates, culture positive rates, and pathogen antimicrobial resistance patterns. Results: Of 6,718 total urine cultures collected, 68% were positive for potentially pathogenic bacteria. During the intervention, significant reductions in the urine culture and positive culture rates were observed (P = .014). Most of the identified potentially uropathogenic isolates were Escherichia coli (38%), Proteus spp (13%), and Klebsiella pneumoniae (12%). A significant decrease was observed during the intervention period in nitrofurantoin resistance among E. coli (P <= .001) and ciprofloxacin resistance among Proteus spp (P <= .001); however carbapenem resistance increased for Proteus spp (P <= .001). Multidrug resistance also increased for Proteus spp compared to the baseline. The high baseline resistance of E. coli to the commonly prescribed antimicrobials ciprofloxacin and trimethoprim-sulfamethoxazole (TMP/SMX) did not change during the intervention. Conclusions: The antimicrobial stewardship intervention program significantly reduced urine culture and culture-positive rates. Overall, very high proportions of antimicrobial resistance were observed among common pathogens; however, antimicrobial resistance trended downward but reductions were too small and scattered to conclude that the intervention significantly changed antimicrobial resistance. Longer intervention periods may be needed to effect change in resistance patterns.