Abstract: Objective　To explore the diagnostic value and advantage of metagenomic next-generation sequencing (mNGS) in the diagnosis of Strongyloidiasis co-infected with other pathogens. Methods　According to the inclusion and exclusion criteria, the clinical data of 5 patients diagnosed with Strongyloides stercoralis in Sun Yat-sen Memorial Hospital, Sun Yat-sen University from November 2020 to October 2022 were retrospectively collected. The clinical characteristics of the patients were analyzed, and the positive rates of Strongyloides stercoralis in 6 samples from 5 patients were compared by using mNGS and liquid-based cytology microscopy, and the detection rates of Strongyloides stercoralis eggs in the feces of 5 patients were compared by using the fecal direct smear method. The consistency between mNGS and bacterial and fungal culture results of the 6 samples was analyzed and compared. Results　Three of the 5 patients were farmers. All 5 patients had underlying diseases, with fever and cough as the main clinical manifestations, and the imaging features were mostly glass shadow and density shadow. Blood routine examination showed an increase in the percentage of neutrophils in 4 of the 5 patients, and none of the 5 patients had an increase in eosinophils. Procalcitonin levels were elevated in all five patients. Among the 6 samples from the 5 patients, the detection rate of Strongyloides stercoralis detection by mNGS was 100%, and the detection rate of liquid-based cytology was 50%. The detection rate of Strongyloides stercoralis eggs using the direct fecal smear method was 20% in 5 patients. Among the 6 samples, Human betaherpesvirus 5 was detected by mNGS, and Pneumocystis jirovecii was detected in 3 samples, Aspergillus fumigates was detected in 3 samples, and Pseudomonas aeruginosa was detected in 2 samples. Except for Strongyloides stercoralis and viruses detected by mNGS, the results of routine culture were completely consistent with those of bacteria and fungi detected by mNGS in 1 sample, inconsistent in 2 samples, and partially consistent in 3 samples. Conclusions　The detection rate of Strongyloides stercoralis by mNGS may be higher than that by fecal direct smear and liquid-based cytology. Compared to conventional bacterial and fungal cultures, mNGS has a wide detection range and may be more suitable for the detection of Strongyloides stercoralis infection co-infected with other pathogens. It presents significant diagnostic advantages for mixed infections and may be used as an early diagnosis method for Strongyloides stercoralis infection.