To investigate the chemical biodiversity of biologically active cyclic hexapeptides in the metabolites from microorganisms, the fermentation broth of Streptomyces alboflavus 313 was analyzed using HPLC, equipped with a diode array detector (DAD), coupled with ESI tandem MS (HPLC-DAD-ESI-MS/MS). In the mass spectra of cyclic hexapeptides, predominant ions [M+H](+), as well as [M-18+H](+), [M-28+H](+) and [M+Na](+), were observed and used to determine the molecular masses, while fragmentation reactions of [M+H](+) were recorded to provide information on the contents of amino acids and their linkage sequence. Based on the fragmentation patterns and comparison with standards, 15 pyrroloindoline-containing natural cyclic hexapeptides, cp01-15, were identified from the microorganism and six of these are reported for the first time.