Extracts from the June collection of aerial parts of Helianthus salicifolius A. Dietr and Helianthus tuberosus L. were obtained using carbon dioxide supercritical fluid extraction with water as co-solvent. The antimicrobial effect in vitro of these extracts was then determined against reference species of bacteria, as well as against fungi (represented by Candida spp.). Both extracts were found to possess antimicrobial activity, with MIC = 0.62–5 mg mL−1 for bacteria and MIC = 5–10 mg mL−1 for yeasts, and both extracts demonstrated suitable bactericidal and fungicidal effect. The highest activity was observed against S. aureus ATCC 29213 (MIC = 0.62 mg mL−1 for H. salicifolius extract; MIC = 2.5 mg mL−1 for H. tuberosus extract) as confirmed by time–kill assay. Higher antioxidant activity was found for H. tuberosus extract (EC50 = 0.332 mg mL−1) as compared to that of H. salicifolius (EC50 = 0.609 mg mL−1). The total polyphenol content (TPC) expressed as gallic acid equivalents (GAE) was 13.75 ± 0.50 mg GAE g−1 of H. salicifolius extract and 33.06 ± 0.80 mg GAE g−1 of H. tuberosus extract. There was a relationship between the antioxidant potential of both extracts and TPC, but not between antistaphylococcal activity and TPC. The ATIR–FTIR spectra of both extracts showed similar main vibrations of the functional groups typical for phytoconstituents possessing bioactivity. The obtained data suggest potential application of these extracts as natural antioxidants and preparations with biocidal activity. Additionally, both extracts may be regarded as potential natural conservants in cosmetics, as well as natural preservatives in food.