Zelkovamycins F and G (<b>1</b> and <b>2</b>), two new natural cyclic octapeptides possessing the unprecedented nonproteinogenic amino acid residues l-α-methyl-threonine and l-α-methyl-<i>allo</i>-threonine, respectively, along with four new analogues, zelkovamycins H-K (<b>3</b>-<b>6</b>), were identified from the endophytic <i>Kitasatospora</i> sp. CPCC 204717. Their structures were elucidated by detailed analysis of NMR and HRESIMS/MS spectroscopic data. The configurations of amino acid residues were determined by Marfey's analysis combined with NMR calculations. Compounds <b>1</b>, <b>2</b>, and <b>4</b> showed potent antibacterial activity against methicillin-resistant <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i>. The structure-activity relationship study revealed that the 2-methyl-3-oxobutyrine and sarcosine residues played important roles in their antibacterial activities. Zelkovamycin (<b>7</b>) and zelkovamycin E (<b>8</b>) exhibited significant antiviral activity against the hepatitis C virus.