BackgroundThe contribution of bronchoalveolar lavage fluid (BALF) microbiota and mycobiota to silicosis has recently been noticed. However, many confounding factors can influence the accuracy of BALF microbiota and mycobiota studies, resulting in inconsistencies in the published results. In this cross-sectional study, we systematically investigated the effects of “sampling in different rounds of BALF” on its microbiota and mycobiota. We further explored the relationship between silicosis fatigue and the microbiota and mycobiota.MethodsAfter obtaining approval from the ethics board, we collected 100 BALF samples from 10 patients with silicosis. Demographic data, clinical information, and blood test results were also collected from each patient. The characteristics of the microbiota and mycobiota were defined using next-generation sequencing. However, no non-silicosis referent group was examined, which was a major limitation of this study.ResultsOur analysis indicated that subsampling from different rounds of BALF did not affect the alpha- and beta-diversities of microbial and fungal communities when the centrifuged BALF sediment was sufficient for DNA extraction. In contrast, fatigue status significantly influenced the beta-diversity of microbes and fungi (Principal Coordinates Analysis,P= 0.001;P= 0.002). The abundance ofVibrioalone could distinguish silicosis patients with fatigue from those without fatigue (area under the curve = 0.938, 95% confidence interval [CI] 0.870–1.000). Significant correlations were found betweenVibrioand haemoglobin levels (P< 0.001, ρ = -0.64).ConclusionsSampling in different rounds of BALF showed minimal effect on BALF microbial and fungal diversities; the first round of BALF collection was recommended for microbial and fungal analyses for convenience. In addition,Vibriomay be a potential biomarker for silicosis fatigue screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12995-023-00377-3.