The MYB family constitutes one of the largest transcription factor families in plants, involving in plant growth and development, signal transduction, and secondary metabolism. Based on our previous 2+3 transcriptome data of Ocimum basilicum var. pilosum, we systematically identified the O. basilicum var. pilosum MYB (ObMYB) family through bioinformatics approaches and explored members associated with essential oil accumulation. Firstly, ObMYB members were screened by homologous alignment of MYB conserved domains, and then their complete open reading frames were predicted and the basic physicochemical properties of these encoded proteins were analyzed. Phylogenetic relationship was reconstructed by neighbor-joining method of MEGA 7.0, while conserved motifs and domains were annotated using MEME and Batch CD-Search. Subsequently, incorporating the previous miRNA data of O. basilicum var. pilosum, the targeted miRNA complementing the ObMYB were predicted by TargetFinder. Lastly, differential expression analysis and visualization analysis of heat maps were conducted by TBtools. Based on the essential oil data from different developmental stages of stems and leaves, the correlation analysis between differential gene expression levels and essential oil content was conducted using SPSS 25 to identify the potential ObMYB members involving in the accumulation of essential oil. The protein-protein interaction (PPI) network was constructed by STRING database for analyzing potential metabolic pathways involved. Research results revealed that a total of 77 ObMYB members were characterized, named as ObMYB-1 to ObMYB-77. Phylogenetic analysis divided them into eight subfamilies (classⅠ-Ⅷ), each comprising 6–14 members. These ObMYB proteins exhibited hydrophilic properties and structural stability, average isoelectric point=7.11 and average hydrophobicity=–0.69. Five miRNAs were found to target and regulate four ObMYB genes. Among these 43 differentially expressed ObMYB genes, ObMYB-4, -9, -27, -34, -42, -46 and -69 displayed strong correlations with essential oil accumulation. Moreover, PPI network analysis indicated the potential involvement of ObMYB-4, -9, -34, -36, -46 and -69 in flavonoid biosynthesis and secondary metabolite regulation. This study systematically identified the ObMYB family members and analyzed their relationship with essential oil accumulation, revealing multiple ObMYB genes significantly associated with the synthesis and accumulation of essential oils. These findings provide a significant theoretical basis for an in-depth understanding of the metabolic regulation mechanisms of O. basilicum var. pilosum essential oil, and to offer new targets and insights for research on plant secondary metabolism and the improvement of aromatic plant quality.