Neem (Azadirachta indica) is a very popular traditional medicinal plant used since ancient times to treat numerous ailments. MicroRNAs (miRNAs) are highly conserved, non-coding, short RNA molecules that play important regulatory roles in plant development and metabolism. In this study, deploying a high stringent genome-wide computational-based approach and following a set of strict filtering norms a total of 44 potential conserved neem miRNAs belonging to 21 families and their corresponding 48 potential target transcripts were identified. Important targets include Squamosa promoter binding protein-like proteins, NAC, Scarecrow proteins, Auxin response factor, and F-box proteins. A biological network has also been developed to understand the miRNA-mediated gene regulation using the minimum free energy (MFE) values of the miRNA-target interaction. Moreover, six selected miRNAs were reported to be involved in secondary metabolism in other plant species (miR156a, miR156l, miR160, miR164, miR171, miR395) were validated by qPCR and their tissue-specific differential expression pattern was observed in leaves and stem. Except for ain-miR395, all the other miRNAs were found overexpressed in the stem as compared to leaves. To the best of our knowledge, this is the first report of neem miRNAs and we believe the finding of the present study will be useful for the functional genomic study of medicinal plants.