Longicatenamides A-D, two diastereomeric pairs of new cyclic hexapeptides, were isolated from the combined-culture of Streptomyces sp. KUSC_F05 and Tsukamurella pulmonis TP-B0596. Their planar structures were determined by spectroscopic analysis including extensive 2D NMR and MS analysis. The absolute configurations of their component amino acids were determined by the use of highly sensitive reagents we recently developed; the highly sensitive-advanced Marfey's method (HS-advanced Marfey's method), which led us to reduce the sample loss and prevent incorrect structural determination. Particularly, the C(beta)-stereochemistry of hyGlu in longicatenamides A and C was assigned without any use of C(beta)-Marfey's methods. Longicatenamide A exhibited weak but preferential antimicrobial activity against Bacillus subtilis.