LC-MS and NMR spectroscopy guided metabolic profiling and dereplication of a crude extract obtained from the fermentation of a marine-derived bacterium, Bacillus subtilis, followed by chromatographic isolation yielded two new cyclic-lipotetrapeptides, bacilotetrins A (1) and B (2). Based on extensive 1D and 2D NMR and high-resolution ESIMS data analysis, the structures of 1 and 2 were elucidated, revealing the unique structures of these lipopeptides consisting of three leucines and a glutamic acid residue cyclized with a lipophilic 3-hydroxy fatty acid. The absolute stereochemistries at selected stereocenters in 1 and 2 were assigned by chemical derivatization and comparison to literature data. Compounds 1 and 2 exhibited anti-MRSA activity with MIC values of 8 to 32 μg/mL. However, these compounds showed no cytotoxicity when tested against prostate and liver cancer cell lines using the standard SRB assay.