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Protothecal mastitis poses an emergent animal health problem in dairy herds, with a high impact on dairy industries, causing heavy economic losses. Current methods of treating protothecal infections are ineffective, and no drug is licensed for use in cattle. The aim of the present study was to check the antialgal activity of 30 chemically defined essential oils (EOs) against Prototheca zopfii and Prototheca blaschkeae isolated from the milk of dairy cows with mastitis. A microdilution test was carried out to estimate the antialgal effectiveness of the selected chemically defined EOs. The microdilution test showed different degrees of inhibition among the examined Prototheca species. The activity of some of the examined EOs seem interesting. In particular, Citrus paradisi yielded the lowest minimal inhibitory concentration values (0.75%) for both algal species. P. zopfii appeared to be more sensitive to EOs in comparison to P. blaschkeae. The present study investigated the in vitro susceptibility of P. zopfii and P. blaschkeae to a wide range of EOs, obtained from different botanical families. Further investigations are necessary to evaluate the efficacy of EO-based formulations intended for the disinfection of both udder and milking products.