Human respiratory syncytial virus (RSV) is one of the predominant pathogens causing lower respiratory tract infection in infants and young children worldwide, whereas there is so far no vaccine or drug against RSV infection for clinical use. In this work, we developed and validated a fluorescence-based high-throughput screening (HTS) assay to identify compounds active against RSV, using RSV-mGFP, a recombinant RSV encoding enhanced green fluorescent protein (EGFP). Thereafter, among 54,800 compounds used for our screen, we obtained 62 compounds active against RSV. Among these hits, azathioprine (AZA) and 6-mercaptopurine (6-MP) were identified as RSV inhibitors with half maximal inhibitory concentration (IC₅₀) values of 6.69?±?1.41 and 3.13?±?0.98?μM, respectively. Further experiments revealed that they functioned by targeting virus transcription or/and genome replication. In conclusion, the established HTS assay is suitable to screen anti-RSV compounds, and the screened two hits of AZA and 6-MP, as potential anti-RSV agents targeting RSV genome replication/transcription, are worthy of further investigation on their anti-RSV activity in vivo.