The genus Vincetoxicum belongs to the family Apocynaceae, known for its medicinal properties. This study focuses on fatty acid, amino acid, sterol, total phenolic, and total flavonoid contents; and antioxidant (by 2,-2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzthioazoline-6-sulfonic acid (ABTS), Cupric ion reducing antioxidant capacity (CUPRAC) tests), antimicrobial, and antibiofilm potential of Vincetoxicum hirundinaria seeds growing wild in Turkey. Chromatographic methods were used for chemical analyses. The most abundant fatty acids were linoleic, oleic, and palmitic acids (48.56%, 28.66%, and 10.15%, respectively). β-sitosterol was the major sterol (61.93%) and glutamic acid was the major amino acid (7463 mg/100 g) in seeds. The total phenolic and flavonoid contents were 12.7 ± 0.28 mg GAE/g seed and 2.55 ± 0.12 mg QE/g seed, respectively. The DPPH radical scavenging activity (0.83 ± 0.02 mM AAE/g seed), ABTS assay (3.61 ± 0.001 mM TE/g seed), and CUPRAC assay (23.8 ± 0.002 mM AAE/g seed) results were evaluated. The MIC values of the tested extract were ranging from 125 to 62.5 µg/mL. The results of the biofilm prevention study were determined as 40% and 37% at the concentrations of MIC/2 and MIC/4, respectively. The results of the biofilm eradication study were determined as 38%, 47%, and 52% at the concentrations of MIC/2, MIC, and 2XMIC, respectively. This is the first study on the seeds’ chemical content and antioxidant potential. The seeds can be viewed as a promising bio-resource for food, pharmaceutical, and biomedical industries due to their chemical, antioxidant, antimicrobial, and antibiofilm properties.