OBJECTIVES: This study aims to characterize the bacterial co-occurrence features and potential interactions associated with the presence of sinus tracts in apical periodontitis in a Chinese population by using 16S rRNA next-generation sequencing (NGS). METHODS: Thirty-one samples from twenty-six patients were collected from root canals. Following the extraction of the bacterial DNA, the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced. Compositional diversity, prominent taxa and co-occurrence network analysis were compared according to the presence or absence of sinus tracts. RESULTS: The overall microbiota in two groups exhibited distinguished patterns. Actinomyces dominated in samples with sinus tracts while Prevotella was the most abundant in samples without sinus tracts. The major pathogens in sinus tracts exhibited a complex co-occurrence network, in which Pseudomonas formed a distinctive cluster with enriched abundance, and the extensive correlations centered on Desulfovibrio and Pseudoramibacter may suggest novel dependencies. In the network without sinus tracts, the Bacteroidetes and Firmicutes taxa presented close internal associations. CONCLUSIONS: The sequencing results confirmed the complexity of the microbiota in AP. The presence of sinus tracts was associated with distinctive infective patterns and complicated microbial co-infection interaction networks. Further investigations should be adopted to elucidate the relationship between the novel interactions and disease progression. CLINICAL SIGNIFICANCE: Exploring the microbial interactions leads to a better understanding of etiology of apical periodontitis. Utilizing next generation sequencing techniques, our research uncovered the bacterial community structure and observed co-infection networks associated with sinus tracts, providing potential insights for prognosis prediction and targeted therapeutics of persistent inflammation.