The objective of this thesis is to test an acute toxicity of special essential oils and other volatile plant compounds to Apis mellifera (honeybee). These essential oils (EOs) and other volatile plant compounds were successfully tested as active inhibitors of honey bee pathogen Ascosphaera apis. This microscopic fungus causes a Chalkbrood disease, a disease which affects the youngest stadium of Apis mellifera larvae and which is, simultaneously with parasitic mites Varroa destructor and another microscopic fungus Nosema apis, the most common disease of Apis mellifera. EOs and compounds with the lowest acute toxicity to Apis mellifera were selected as the result of this thesis. Because of their inhibition effect to Ascosphaera apis, they could be recommended as an alternative cure against Chalkbrood disease in real beekeeping practise. A whole process of testing was divided into two parts. At first, the acute toxicity of a wider set of EOs and compounds was tested to adult individuals of Apis mellifera. They were placed in Petri dishes and completely exposed to test compounds. The exposure was realized by whole individual’s bodies. The objective of this part was to select a smaller set of compounds with lower acute toxicity and to recommend them for more detailed tests. In the second part, the recommended EOs and compounds were tested again in conditions simulating a real environment of honeybee hives. A new method of potential application of volatile substances in a real practise was tested too. This method is based on volatilizing of substances to a stream of warm air (35 °C) and subsequently on the exposure of Apis mellifera adult individuals to this air stream containing test substances in a vapour phase with specific concentration. For simulating of a real environment in hives, special cages (commonly used in apiary tests) and a thermobox with a closed circulation of air were used. The temperature in the thermobox was 35 °C constantly – the temperature which is common in hives on honeycombs with larvae and pupae. The result of the first part of tests was a selection of the following substances - carvacrol, geraniol, citral as the pure volatile plant compounds and one essential oil (a mixture of more compounds) from Cymbopogon citratus. While these substances were tested in the second part of the tests, the lowest acute toxicity was found for the compound citral. This pure plant volatile compound was recommended for a next research for potential usage as an alternative cure against Ascosphaera apis.