Background: Although urinary tract infections (UTIs) are one of the common infections, especially among elderly, the gold standard diagnostic method is still bacterial culture. Since large percentages of urine culture are negative, it seems that specialists should pay more attention to other bacteria, such as fastidious bacteria. The aim of this study was to use the method of fastidious bacteria isolation for urine samples of elderly females with negative results for conventional culture. Methods: This study was conducted at Imam Khomeini hospital and fastidious bacteria from urine samples of 1075 females older than 60 years were isolated and identified. Developing changes in culture media and incubation conditions and gas levels led to isolation of fastidious bacteria. Results: After overnight aerobic culturing of 1075 urine samples, 383 cases had positive, 99 cases had low counting, 159 cases had mixed, and 434 cases had negative test results. Among 434 negative cases obtained from these methods, and using fastidious bacteria in this study, 147 subjects had Fastidious bacterial, more than 50% of which was associated with infection symptoms, indicating a significant difference. Conclusions: For the isolation and detection of urinary tract infections in elderly female patients with negative aerobic and night culture results, especially if it was associated with the presence of symptoms, culturing the samples in additional and selective environments with changes in the incubation conditions is necessary in order to isolate bacteria.