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Seeds of <i>Vitis vinifera</i> L. with a high content of bioactive compounds are valuable by-products from grape processing. However, little is known about the bioactivity of seeds from other <i>Vitis</i> species. The aim of this study has been to compare the phenolic composition, antimicrobial activity, and antioxidant activity of extracts from seeds of four <i>Vitis</i> species (<i>V. riparia</i> Michx., <i>V. californica</i> Benth., <i>V. amurensis</i> Rupr., and <i>V. vinifera</i> L.). Antioxidant activities were assessed as ferric-reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•) scavenging activity, and oxygen radical absorbance capacity (ORAC). The antimicrobial activity was determined using the microdilution method against some Gram-negative (<i>Escherichia coli</i>, <i>Salmonella enterica</i> ser. Typhimurium, and <i>Enterobacter aerogenes</i>) and Gram-positive (<i>Enterococcus faecalis</i> and <i>Staphylococcus aureus</i>) bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to evaluate the phenolic profile of extracts. Flavan-3-ols, procyanidins, phenolic acids, flavonols, anthocyanins, and stilbenoids were detected. (+)-Catechin and (−)-epicatechin turned out to be the most abundant in the phenolic profile of <i>V. amurensis</i> seed extract. Phenolic acids prevailed in the extract from <i>V. vinifera</i> seeds. The <i>V. riparia</i> and <i>V. californica</i> seed extracts had higher contents of most individual phenolics compared to the other <i>Vitis</i> species. They also showed a higher total phenolic content, DPPH^• scavenging activity, ORAC, and overall antibacterial activity. Total phenolic content significantly correlated with antioxidant activity and antimicrobial activity against <i>E. coli</i>. The principal component analysis (PCA) showed discrimination between <i>V. vinifera</i>, <i>V. amurensis</i>, and clustered <i>V. riparia</i> and <i>V. californica</i> with respect to variables. To recapitulate, this research demonstrates that seeds of different <i>Vitis</i> species, especially <i>V. riparia</i> and <i>V. californica</i>, are sources of molecules with antioxidant and antimicrobial activities that can be used in different sectors, such as in the food, cosmetic, and pharmaceutical industries.