Three new dihydroisocoumarin glycosides (iso-scorzopygmaecoside, scorzoaucherioside I, and scorzoaucherioside II) together with five known compounds (scorzopygmaecoside, scorzocreticoside II, 3,5-O-dicaffeoyl-epiquinic acid, 3,5-O-dicaffeoylquinic acid, and 3,4-dihydroxyphenyl caffeate) were isolated from the aerial parts of the Scorzonera aucheriana collected from Sivas province in Turkey. The isolated compounds' structures were elucidated based on NMR (1H, 13C/APT, COSY, HMBC, HSQC, and TOCSY), UV, FT-IR, and LC-QTOF-MS data. These compounds have been isolated for the first time from S. aucheriana. An antimicrobial assay against ten microorganisms was applied to all isolated compounds. Iso-scorzopygmaecoside, scorzoaucherioside I, and 3,4dihydroxyphenyl caffeate showed notable anti-pseudomonal activity. Iso-scorzopygmaecoside and scorzoaucherioside II exhibited strong activity against Mycobacterium smegmatis with MIC values of 25.6 mu g/mL and 21.2 mu g/mL, respectively.