In this study, quantitative PCR (qPCR) and high-throughput sequencing were used to simultaneously examine both bacteria and fungi across temporal and spatial scales in activated sludge from wastewater treatment plants (WWTPs). The ratio of fungi to bacteria was 0.43% on average after accounting for the multicopies in 16S rRNA gene (54.63%), indicating the number of fungi was far lower than bacteria in active sludge. The Miseq sequencing results revealed obvious seasonal and spatial variations in bacterial and fungal distribution patterns in WWTPs. Compared to bacteria, fungi showed a lower divergence in alpha and beta diversity, and exhibited less taxonomic diversity in both abundant and rare subcommunities at the class level, suggesting that the fungal community was less variable in this artificial ecosystem. Such variation of microbial communities was significantly correlated with geographical distance, DO, temperature, HRT, SRT, COD, TN and TP. In activated sludge, the main function of bacteria was chemoheterotrophy, fermentation, and nitrogen cycling processes, while the dominant functional guilds of fungi were saprotroph, animal pathogen, and animal endosymbiont. Moreover, both bacteria and fungi could play important roles in the degradation of toxicants, like hydrocarbon and aromatic compounds. Display omitted •In activated sludge samples, fungal to bacteria ratio was less than 1%.•Seasonal and spatial variations were observed in bacterial and fungal communities.•Fungi exhibited lower divergence in taxonomy, alpha and beta diversities than bacteria.•Bacteria and fungi owned different functions but worked jointly in activated sludge.