Genome mining and biocatalytic modification of chemical structures are critical methods to develop new antibiotics. In this study, eight new fasamycins (<b>3</b>, <b>4</b>, <b>6</b>, and <b>8</b>-<b>12</b>) along with five known analogues (<b>1</b>, <b>2</b>, <b>5</b>, <b>7</b>, and <b>13</b>) were obtained by the overexpression of two phosphopantetheinyl transferases (PPtases) in <i>Streptomyces kanamyceticus</i> and biocatalytic transformation with two halogenases. These new compounds displayed significant activity against <i>Staphylococcus aureus</i> and <i>Bacillus subtilis</i>, in particular, C-29-methyl and C-2/C-22-halogen derivatives. This study increases the chemical diversity of bioactive fasamycin derivatives and provides useful halogenation tools for engineering their scaffolds.