In the course of screening for inhibitors against reverse transcriptase of avian myeloblastosis virus, two inhibitors were isolated from the cultured broth of Streptomyces retrostaticus: one was identified as streptonigrin (1), an aminoquinolinequinoide antibiotic with strong antitumor activity but limited clinical application due to severe side effects (mainly bone marrow depression), and the other was a novel compound named retrostatin. Chemical modifications of the amino group on C7/C5' or the hydroxyl group on C8' of 1 usually led to substantial loss of antitumor activity; however, streptonigrin methyl ester (2) retained marked antitumor activity and had a maximum tolerated dose in humans 5 to 6 times that of 1. Therefore, the carboxyl group on C2' of 1 was modified via Method A (acid chloride method using 1's acid chloride as an intermediate) or Method B (reaction with amines in the presence of phenyl bis(2-thioxo-1,3-thiazolidine-3-yl)phosphine oxide) to synthesize derivatives 2-13. This study investigated the biological properties (antimicrobial and cytocidal activities) of 1 and its derivatives. Antimicrobial tests (agar dilution method) showed that streptonigrin hydroxamic acid (8) and streptonigrin hydrazide (9) had weaker activity than 1, while other derivatives exhibited very weak or no activity. Cytotoxicity assays against tumor cells (L5178Y/S, L5178Y/ADR, P388/S, P388/ADR) revealed that 1, 2, and 9 had 32, 8, and 8 times stronger cytotoxicity than adriamycin (ADM) against L5178Y/S; notably, the ADM-resistant subline L5178Y/ADR was more sensitive to these compounds. Additionally, P388/ADR (an ADM-resistant subline of P388 leukemia) showed no significant cross-resistance to 1, 2, or 9.