Rakicidins G-I (1e3), three new cyclic depsipeptides including a long aliphatic chain without terminal methyl branching were isolated from culture broth of Micromonospora chalcea FIM 02e523, along with rakicidin E (4). Their structures were elucidated by extensive NMR and HR-ESI-MS analyses. Compounds 1e4 were evaluated for their cytotoxic activities against HCT-8 and PANC-1 human tumor cell lines under the hypoxic and normoxic conditions. They were approximately 18.2e20.3-fold more cytotoxic under hypoxic than under normoxic conditions on PANC-1, and compared with 7.4e8.7-fold more cytotoxic under hypoxic than under normoxic conditions on HCT-8. Meanwhile, compounds 1e3 were also tested inhibitory activities against gram positive anaerobic bacteria and the results exhibited MIC values in the range of 0.125e8 mg/mL.