BACKGROUND: Mycoplasma hominis (Mh) is a conditional pathogenic microorganism in humans, typically colonizing the genitourinary tract of adults and causing infections in this system. However, Mh is rarely reported as a causative agent of peritoneal dialysis (PD)-associated peritonitis. CASE PRESENTATION: A 46-year-old woman undergoing maintenance PD presented with abdominal pain and cloudy PD effluent. Her PD modality is continuous ambulatory peritoneal dialysis (CAPD). Cultures of the PD effluent and blood were negative, and empirical antibiotic therapy was ineffective, leading to a diagnosis of refractory peritonitis. Metagenomic next-generation sequencing (mNGS) detected Mh in the PD effluent. Treatment was adjusted to intraperitoneal (IP) clindamycin combined with intravenous drip(ivdrip) moxifloxacin, resulting in complete recovery. CONCLUSION: Patients with culture-negative PD-associated peritonitis or refractory peritonitis should be evaluated for potential Mh infection. mNGS enables rapid detection of pathogens that traditional methods may fail to identify. A combination of moxifloxacin and clindamycin is effective for treating PD-associated peritonitis caused by Mh.