Determining the benign or malignant status of indeterminate pulmonary nodules (IPN) with intermediate malignancy risk is a significant clinical challenge. Oral microbiota-lung cancer interactions have qualified oral microbiota as a promising non-invasive predictive biomarker in IPN.Prospectively collected saliva, throat swabs, and tongue coating samples from 1040 IPN patients and 70 healthy controls across three hospitals. Following up, the IPNs were diagnosed as benign (BPN) or malignant pulmonary nodules (MPN). Through 16S rRNA sequencing, bioinformatics analysis, fluorescence in situ hybridization (FISH), and seven machine learning algorithms (support vector machine, logistic regression, naïve bayes, multi-layer perceptron, random forest, gradient-boosting decision tree, and LightGBM), we revealed the oral microbiota characteristics at different stages of HC-BPN-MPN, identified the sample types with the highest predictive potential, constructed and evaluated the optimal MPN prediction model for predictive efficacy, and determined microbial biomarkers. Additionally, based on the SHAP algorithm interpretation of the ML model's output, we have developed a visualized IPN risk prediction system on the web.Saliva, tongue coating, and throat swab microbiotas exhibit site-specific characteristics, with saliva microbiota being the optimal sample type for disease prediction. The saliva-LightGBM model demonstrated the best predictive performance (AUC = 0.887, 95%CI: 0.865-0.918), and identified Actinomyces, Rothia, Streptococcus, Prevotella, Porphyromonas, and Veillonella as biomarkers for predicting MPN. FISH was used to confirm the presence of a microbiota within tumors, and external data from a lung cancer cohort, along with three non-IPN disease cohorts, were employed to validate the specificity of the microbial biomarkers. Notably, coabundance analysis of the ecological network revealed that microbial biomarkers exhibit richer interspecies connections within the MPN, which may contribute to the pathogenesis of MPN.This study presents a new predictive strategy for the clinic to determine MPNs from BPNs, which aids in the surgical decision-making for IPN.Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.