Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of infectious mortality worldwide, and the emergence of drug-resistant strains has intensified the search for new therapeutic agents. Marine macroalgae from underexplored ecosystems such as Antarctica represent promising sources of bioactive metabolites. Here, we investigated the antimycobacterial and anti-inflammatory potential of Desmarestia anceps. Crude extracts from eight sites across the Antarctic Peninsula were screened, among which the extract from Penguin Island exhibited the strongest effect against Mtb H37Rv (MIC₅₀ = 6.6 µg/mL) and concomitant reduction of nitric oxide (IC₅₀ = 4.2 ± 1.1 µg/mL) and TNF-α production (IC₅₀ = 21.4 ± 1.1 µg/mL). Bioassay-guided fractionation identified DAF2 as the most promising, displaying potent antimycobacterial effects (MIC₅₀ = 16.9 µg/mL against H37Rv), no cytotoxicity in murine macrophages (CC₅₀ > 100 µg/mL), and suppression of NO levels in LPS-stimulated macrophages, thus suggesting a dual role in infection control and inflammation modulation. High-speed counter-current chromatography (HSCCC) of DAF2 revealed sterols, fatty acids and diterpenoids as major constituents, among which saringosterol—previously shown to have potent antimycobacterial activity—and fucosterol were prominent. Fucosterol, isolated and tested individually, exhibited remarkable activity against Mtb H37Rv (MIC₅₀ = 0.37 ± 0.09 µg/mL) and the hypervirulent strain M299 (MIC₅₀ = 3.05 ± 1.11 µg/mL), with a selectivity index above 10 based on macrophage cytotoxicity (CC₅₀ = 51.45 µg/mL). Altogether, these findings highlight D. anceps as a promising source of natural products with combined antimycobacterial and anti-inflammatory activities, positioning fucosterol as a promising lead for TB drug discovery.