<p><i>Mycobacterium marinum</i> is one of the main pathogens in aquaculture with direct or indirect impacts on human, animal, and environmental health, significantly slowing progress in productive and economic sectors. Microalgae are a potential source of new antimicrobial compounds; however, they are still little explored. In the present study, we propose the use of an alternative culture medium to produce bioactive compounds from <i>Conticribra weissflogii</i> against the important mycobacterial pathogen <i>M. marinum.</i> Complementary tools, ¹H nuclear magnetic resonance and high-performance liquid chromatography, were employed to characterize the major compounds present in the bioactive extracts. The nonpolar extracts were rich in fatty acids and pigments, such as fucoxanthin, and exhibited potent antimycobacterial activity. The hexanic and ethyl acetate extracts from <i>C. weissflogii</i> were effective against <i>M. marinum</i> with a minimum inhibitory concentration of 0.1&#xa0;mg·mL⁻¹ and low cytotoxicity. This pathogen poses a threat to human society, affecting aquaculture production, leading to alarming economic losses, contaminating natural resources, and increasing the spread of zoonotic diseases with high morbidity and mortality. Addressing this issue through a One Health approach is necessary. In this context, the use of natural products with antimicrobial properties, such as the diatomaceous extract studied here, may be an effective solution.</p> Graphical Abstract <p></p>