Abstract In order to find out whether the chemotherapeutic properties of bisazo dyes derived from bis(4‐aminophenyl)‐sulfone are due to the dyes themselves or to the amine tetrazotized (reduction product of the dye), thirty bisazo dyes were prepared by condensing tetrazotized bis(4‐aminophenyl)‐sulfone with various types of coupling components. The in vitro activity of these dyes against M. Tb. was studied. The most active ones were those derived from 1,3‐diaminobenzene, meta‐aminobenzoic acid, salicylic acid, and 8‐hydroxy‐quinoline. They inhibited the growth of this micro‐organism completely at a concentration of 2 mg per 10 cm3 culture. The derivative from meta‐amino‐benzoic acid was still strongly bacteriostatic at a concentration of 0.2 mg per 10 cm3 culture. (3) Nine of the above bisazo dyes representing different types were also tested chemotherapeutically. Only three of them (derivatives from meta‐phenylene diamine, salicylic acid and H acid) were found to be as effective as sulfanilamide in Streptococcus hemolyticus infected mice. The dye from meta‐phenylene diamine was also as effective as sulfadiazine in mice infected with McGovern, normal Type I Pneumococcus. They were ineffective against tetanus toxemia, influenza virus, and rabic virus in mice. Five of these dyes were tested in vitro against ameba. Two of them (derivatives from 3,5‐diamino‐benzoic acid and 2‐methyl‐1,4‐naphthohydroquinone) were as effective as chloroquine standard. Derivatives from para‐amino‐benzoic acid and phloroglucinol were about one half as active, while the dye from H acid was only amebastatic at dilutions between 1: 500 and 1: 5000. The relationship between chemical structure and biological activity of these bisazo dyes was discussed.