Transcriptional control by enhancer elements play an important role in brain development and function, and many variants associated with neurodevelopmental and psychiatric disorders are located in predicted enhancers. However, there is little functional evidence of enhancer activity and limited identification of enhancer’s target genes in brain-derived cells. Here we assessed the function of 979 putative enhancers in primary human astrocytes through CRISPRi screening, combining epigenetic silencing by dCas9-KRAB with scRNA-seq. This screen identified over a hundred active enhancers and uncovered their target genes. We found that enhancer activity is associated with enhancer RNA (eRNA) expression, often doesn’t involve the nearest gene, and most enhancers regulate genes within 200kb. The genes regulated by these enhancers were overrepresented among genes associated with neurodegenerative disorders and glioma. Furthermore, 90% of the regulatory interactions captured by CRISPRi screening were not captured by eQTL data, suggesting that functional characterisation of enhancers allows for a greater understanding of the regulatory landscape of brain cells, with implications for understanding the molecular mechanisms underlying brain disorders.