Sick building syndrome (SBS) is a collection of nonspecific syndromes linked with the built environment. The occurrence of SBS is associated with humidity, ventilation, moulds and microbial compounds exposure. However, no study has reported the association between indoor microbiome and SBS. In this study, 308 students were surveyed for SBS symptoms from 21 classrooms of 7 junior high schools from Johor Bahru, Malaysia, and vacuum dust from floor, desks and chairs was collected. High throughput amplicon sequencing (16S rRNA gene and ITS region) and quantitative PCR were conducted to characterize the absolute concentration of bacteria and fungi taxa. In total, 326 bacterial and 255 fungal genera were detected in dust with large compositional variation among classrooms. Also, half of these samples showed low compositional similarity to microbiome data deposited in the public database. The number of observed OTUs in Gammaproteobacteria was positively associated with SBS (p = 0.004). Eight microbial genera were associated with SBS (p Rhodomicrobium, Scytonema and Microcoleus, were protectively (negatively) associated with ocular and throat symptoms and tiredness, and Izhakiella and an unclassified genus from Euzebyaceae were positively associated with the throat and ocular symptoms. Three fungal genera, Polychaeton, Gympopus and an unclassified genus from Microbotryaceae, were mainly positively associated with tiredness. The associations differed with our previous study in microbial compounds (endotoxin and ergosterol) and SBS in the same population, in which nasal and dermal symptoms were affected. A higher indoor relative humidity and visible dampness or mould in classrooms were associated with a higher concentration of potential risk bacteria and a lower concentration of potential protective bacteria (p