Background <p>Infectious etiologies of deep neck space abscess (DNSA) by conventional culture tests can be challenging, which also leads to frequent irrational antibiotic usage. Metagenomic next-generation sequencing (mNGS), as a novel method for analyzing the complex microbial ecosystem from clinical samples, has been utilized in clinical research and practice of various infectious diseases but deep neck space abscess. We here aimed to explore the clinical value of mNGS for pathogen detection and treatment guidance in DNSA patients compared with conventional culture tests.</p> Methods <p>One hundred six patients diagnosed with DNSA were retrospectively enrolled and allocated into mNGS group and culture group according to whether mNGS was conducted. The pathogen detection effectiveness was of mNGS was compared with conventional culture. Effectiveness of mNGS-modified antimicrobial therapy was evaluated by comparing the treatment outcomes between two groups.</p> Results <p>mNGS showed a significantly higher detection rate than conventional culture (<i>p</i> &lt; 0.05) with faster result acquisition. Treatment success rate of patients in the mNGS group was significantly higher than in the culture group (RR: 1.22, 95%CI: 1.07–1.82, <i>p</i> = 0.033). Besides, patients in the mNGS group had shorter duration of irrational antimicrobial therapy, shorter hospital stay and less medical costs (<i>p</i> &lt; 0.05).</p> Conclusions <p>mNGS is an effective technology for facilitating pathogen detection and improving treatment outcomes of DNSA patients.</p>