Penicillium marneffei is a rare invasive fungal infection that primarily occurs in the southeastern and eastern regions of Asia in HIV-positive individuals. Herein we present a rare case of disseminated P. marneffei infection with fungemia and intestinal perforation in an HIV-negative patient in China. Extensive diagnostic evaluations did not reveal the cause of the disease; we use the automated microbiology system, matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), polymerase chain reaction (PCR), and the sequencing analysis to detect and identify the pathogen in positive blood culture of patient. The pathogen was initially misdiagnosed as Stephanoascus ciferrii by automated microbiology system. However the sequencing analysis revealed the pathogen to be P. marneffei. The patient was treated with amphotericin B, and a significant improvement in his condition was achieved. However, after a month, the patient suffered from an intestinal perforation, which may have been caused by P. marneffei. Although P. marneffei has been found in select regions, and appears mostly in patients with a weak immune system, particularly HIV-positive individuals. It is important to note that in addition to patients who are HIV-positive, anyone with a weak immune system can be infected with P. marneffei. DNA sequencing is generally considered to be the gold standard for the identification of microorganisms.