The presence of erythromycin, an antibiotic commonly used to treat bacterial infections, at high concentrations in freshwater bodies remains a significant ecological hazard. However, the effects of free erythromycin on microorganisms, especially freshwater biofilms, are still not fully understood with regard to microbial diversity, active constituents and their underlying functional activities. In this study, we used a laboratory-cultured multispecies biofilm model to further unravel the gene expression profiles of freshwater biofilms subjected to sub-inhibitory concentrations of erythromycin via metatranscriptomic analysis. Our metatranscriptomic analysis revealed apparent changes in the relative expression of Proteobacteria, Microsporidia, Bacteroidetes, Actinobacteria, and Planctomycetes after erythromycin exposure. The KEGG pathway and EggNOG analyses suggested that erythromycin induced specific functional activities, such as increased relative expression in translation, transcription, cell motility, cell wall biogenesis, and defense mechanisms. A confocal laser scanning microscopic analysis showed significant structural differences in biofilm roughness, which further validated the above-mentioned observations from the perspective of the biofilm phenotype. Further analysis of specific genes revealed significant increases in the expression of biofilm PGA synthesis, erythromycin-resistant genes, and stress-related genes after erythromycin exposure. Significant changes were observed in the biofilm structure, active microbiome, and functional attributes following biofilm exposure to erythromycin. Expression analysis of specific genes suggested that biofilms actively initiated a defensive system under sub-inhibitory erythromycin pressure. Our findings provide an in-depth overview of how multispecies biofilms respond to erythromycin exposure at the gene expression level.