Mycobacterium abscessus is an emerging pathogen causing severe lung infections in cystic fibrosis patients. This species transitions from a smooth (S) to a rough (R) variant upon the loss of surface glycopeptidolipids (GPL). While the S variant colonizes the airways, the R variant develops during infection and manifests as a more aggressive pathogen associated with declining lung functions. To describe the molecular mechanisms of the S-to-R switch, a thorough structure-activity relationship study was undertaken to decipher the role of GPL glycosylation in morphotype transition and pathogenesis. The absence of the 6-deoxy-talose and the first rhamnose in mutants lacking the glycosyltranferases Gtf1 and Gtf2, respectively, resulted in the S-to-R transition. Strikingly, the gtf1 and gtf2 mutants displayed a strong propensity to form cords and abscesses in zebrafish, leading to robust and lethal infection. This underscores the importance and differential contribution of GPL monosaccharides in virulence and infection outcomes. In addition, we recently discovered a second GPL-related locus comprising MAB_4690c, encoding a non-ribosomal peptide synthetase. A MAB_4690c deletion mutant failed to produce a yet undescribed lipid, designated GL8P, sharing an acylated octapeptide core adorned by mono- or di-O-rhamnosyl substituents. This mutant showed impaired uptake and survival in macrophages and was attenuated in mice. GL8P elicited also a strong humoral response in M. abscessus-infected patients, identifying GL8P as a novel selective marker for future diagnostic developments. Overall, these findings indicate that M. abscessus produces a wide array of surface glycopeptidolipids participating in host cell interactions and virulence.