Mycobacterium species show distinctive characteristics with significant medical implications. Mycobacteria, including Mycobacterium tuberculosis and non-tuberculous mycobacteria, can form biofilms that facilitate their survival in hostile environments and contribute to development of antibiotic resistance and responses by the host immune system. Mycobacterial biofilm development is a complex process involving multiple genetic determinants, notably mmpL genes, which regulate lipid transport and support cell wall integrity, and the groEL gene, which is essential for biofilm maturation. Sliding motility, a passive form of surface movement observed across various mycobacterial species, is closely associated with biofilm formation and colony morphology. The unique sliding motility and biofilm-forming capabilities of Mycobacterium spp. are pivotal for their pathogenicity and persistence in diverse environments. A comprehensive understanding of the regulatory mechanisms governing these processes is crucial for the development of novel therapeutic strategies against mycobacterial infections. This review provides a detailed examination of our current knowledge regarding mycobacterial biofilm formation and motility, with a focus on regulation of these processes, their impact on pathogenicity, and potential avenues for therapeutic intervention. To this end, the potential of natural and synthetic compounds, including nanomaterials, in combating mycobacterial biofilms and inhibiting sliding motility are discussed as well. These compounds offer new avenues for the treatment of drug-resistant mycobacterial infections.