Lobophorins (LOBs) are a growing family of spirotetronate natural products with significant cytotoxicity, anti-inflammatory, and antibacterial activities. Herein, we report the transwell-based discovery of sp. CB09030 from a panel of 16 in-house strains, which has significant anti-mycobacterial activity and produces LOB A (), LOB B (), and LOB H8 (). Genome sequencing and bioinformatic analyses revealed the potential biosynthetic gene cluster (BGC) for -, which is highly homologous with the reported BGCs for LOBs. However, the glycosyltransferase LobG1 in . sp. CB09030 has certain point mutations compared to the reported LobG1. Finally, LOB analogue (O-β-D-kijanosyl-(1→17)-kijanolide) was obtained through an acid-catalyzed hydrolysis of . Compounds - showed different antibacterial activities against and , which revealed the varying roles of different sugars in their antibacterial activities.