The Institute in Prague came under the control of German medical authorities following the occupation of Bohemia in 1939. Prof. Pelc was relieved of his directorial post towards the end of 1940. At the start of 1941, a professor from the German institute of public health, Dr Pesch, was appointed as head; he died in May of the same year. His place was taken by another German national, Doc. Schubert, who had until then been the head of the department for biological control of drugs. In 1942, during the frenzied aftermath following the assassination of Reinhard Heydrich, four doctors were executed – Prof. Pelc, doc. Strimpl, Dr. Bouček, and Dr. Klíma. Some of the employees were also deported to the concentration camps. In the same year a veterinary department was set up, containing laboratories for analysis of meat and milk.

In 1943, there was an increase in the production of sera; the German army was supplied with diphtheria sera and the Behring Company received sera against anthrax. The huge increase in sera production, however, proved to be of little use to the German army. The supplies of tetanus and gangrene sera were used during the May uprising in 1945.

The first post-war director of the NIPH was Prof. Vilém Hons, MD.