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A 59-year-old man developed a high fever, elevated white blood cell count, elevated C-reactive protein level, and perineal pain 5 days after robot-assisted laparoscopic radical prostatectomy. Treatment with cefmetazole was ineffective. A urine specimen was submitted for culture on postoperative day 7, and Mycoplasma hominis (M. hominis) was detected 1 week later. Cefmetazole was therefore switched to quinolone. The clinical symptoms and laboratory data immediately showed marked improvement. M. hominis has been shown to inhabit the genitourinary tract. Although it is considered to induce urethritis, its pathogenicity in healthy male subjects has not been investigated. M. hominis is difficult to detect and is resistant to β-lactams because it lacks a cell wall. Urine culture sometimes results in false-negative results. In cases where empirical therapy for postoperative infection is ineffective, surgeons should recognize the possibility of M. hominis involvement and consider changing the antibiotic used.