Background <p>Microsporidia are genetically diverse, obligate intracellular pathogens that are challenging to diagnose due to their resistance to conventional culture methods. <i>Enterocytozoon bieneusi </i>is a primary cause of microsporidiosis, particularly in immunocompromised individuals. This report details a complex case of mixed bacterial and microsporidial infection in a renal transplant recipient, highlighting the utility of advanced molecular diagnostics.</p> Case presentation <p>A 37-year-old male with a 16-year history of renal transplantation presented with recurrent high fever, chills, and hematochezia. Initial workup revealed severe anemia, thrombocytopenia, and acute kidney injury. Blood cultures grew ESBL-producing <i>Escherichia coli </i>and <i>Klebsiella pneumoniae</i>. CT imaging suggested a duodenobiliary fistula. Due to persistent fever despite broad-spectrum antibiotics, metagenomic next-generation sequencing (mNGS) was performed, identifying <i>E. bieneusi </i>(185 reads) alongside multiple bacteria and viruses. Treatment was adjusted to include meropenem, daptomycin, and albendazole, with careful modulation of immunosuppression. The patient subsequently tested positive for SARS-CoV-2 but showed significant clinical improvement with the adjusted regimen and was discharged after 24 days.</p> Conclusion <p>This case underscores the diagnostic value of mNGS in identifying rare, opportunistic pathogens in immunocompromised hosts. Successful management requires a multidisciplinary approach, combining targeted antimicrobial therapy, immunomodulation, and aggressive supportive care.</p> Clinical trial number <p>Not applicable.</p>