In the course of a screening program for bioactive compounds from a marine natural product library, a newly isolated Actinomycetes strain, designated as MS100061, exhibited strong anti-Mycobacterium bovis Bacillus Calmette-Guérin (BCG) activity. The strain belongs to the genus Streptomyces according to its morphological and 16S rDNA phylogenetic analysis. Bioassay-guided isolation resulted in a new spirotetronate, lobophorin G (1), together with two known compounds, lobophorins A (2) and B (3). The structures were elucidated by extensive spectroscopic methods and comparison with literatures. Compounds 1-3 were subjected to anti-BCG, antituberculosis, and antibacterial screening and exhibited potent anti-BCG activity with minimum inhibitory concentration (MIC) values of 1.56, 1.56, and 0.78 μg/ml, respectively, and moderate anti-Mycobacterium tuberculosis H37Rv activity with MIC values of 32, 32, and 16 μg/ml, respectively. The MIC values of compounds 1-3 against Bacillus subtilis were 3.125, 12.5, and 1.56 μg/ml, respectively, indicating great potential for antibacterial drugs. In addition, this is the first report of the anti-BCG and antituberculosis activities of lobophorins.