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In the last years, the possibility to exploit autochthone microalgae in regional applications has been explored. The regional-based microalgal industry may bring several benefits, as autochthone microalgae are already adapted to the biotic and abiotic stresses of their environment. In this work, this concept was applied to Sicily, in which three microalgal strains were collected from the coastline. Monoalgal strains were then isolated and molecular characterization was performed for the species determination. Three of them, two strains of Chlorella and one of Dunaliella, were cultivated in lab-scale in four different conditions: Low Temperature-Low Salt (LT-LS), High-Temperature-Low Salt (HT-LS), Low Temperature-High Salt (LT-HS) and High Temperature-High Salt (HT-HS) to investigate the role of each condition on the growth performance, the productivity and the biochemical composition of the microalgal biomass. In particular, lipid, fatty acid composition and antioxidant capacity were assessed. Results indicated that one of the Sicilian strains of Chlorella has a better growth performance at a higher temperature while the Dunaliella strain is tolerant to high-salt stress. Moreover, the biochemical composition appears to be strongly influenced by temperature and salt stresses: the lipid content decreased in all the strains and a significant shift in fatty acid composition was observed, with an increase in the content of n-3 PUFAs in some cases. Results indicated that also the carotenoids content decreased in some of the tested stress conditions. The results obtained in this research represent a first step for developing a regional-based microalgal industry in Sicily by exploiting the natural biodiversity of the Sicilian environment.