Antibiotic C-1027, a potent antitumor chromoprotein from Streptomyces globisporus C-1027, consists of an acidic protein and a labile chromophore with a 9-membered 1,5-diyn-3-ene core in a 16-membered macrocyclic ring and benzoxazolinate/aminosugar side chains. During large-scale production for toxicological evaluation and formulation studies, another active chromoprotein (tentatively termed NeoC-1027) was identified. This work describes the isolation, structural elucidation, and biological activities of NeoC-1027, as well as the isolation and structure of its two chromophore components (Chr-I and -II) which can be separated from C-1027's chromophores (Chr-III and -IV) via reversed-phase HPLC of freshly extracted chromophore preparations. NeoC-1027's apoprotein exhibits similar physicochemical properties (amino acid composition, molecular weight, isoelectric point) to that of C-1027. Both compounds show comparable antibacterial activities, with minimum inhibitory concentrations (MICs) of ≤0.05–0.2 μg/ml against Gram-positive bacteria and 0.39–0.78 μg/ml against some Gram-negative bacteria like Escherichia coli. NeoC-1027 displays potent cytotoxicity against KB cells at concentrations less than 0.1 ng/ml and significant inhibition of Colon 26 adenocarcinoma growth (74%) in mice following intravenous administration on days 1, 5, and 9.