In this paper, three extraction methods: conventional hydrodistillation, microwave assisted hydrodistillation and supercritical carbon dioxide extraction, were applied to isolate essential oils from two Eucalyptus species. To our knowledge, it is the first study dealing with extraction and characterization of essential oils from E. intertexta and E. diversifolia from the south of Tunisia. A comparison between species in terms of yield, extraction time, and chemical composition of essential oils as well as their anti-inflammatory and anti-Alzheimer activities was done. The obtained yields varied from 0.88 to 8.35% depending on the species and extraction process. Chemical composition of essential oils was evaluated using gas chromatography and mass spectrometry. Eighty-six compounds were identified, whose major are 1,8-cineole (60.36%), and Î±-pinene (24.72%) in E. diversifolia EOs and 1,8-cineole (33.50%), caryophyllene oxide (21.51%) and p-cymene (21.29%) in E. intertexta EOs. Microwave assisted hydrodistillation was shown to be the faster compared to the other studied methods. Interesting activities were observed with E. diversifolia essential oils extracted by hydrodistillation (50% inhibition in anti-inflammatory test (5-lipoxygenase)) and by microwave assisted hydrodistillation (49% inhibition in anti-Alzheimer test (acetylcholinesterase)).