Seeds ofVitis viniferaL. with a high content of bioactive compounds are valuable by-products from grape processing. However, little is known about the bioactivity of seeds from otherVitisspecies. The aim of this study has been to compare the phenolic composition, antimicrobial activity, and antioxidant activity of extracts from seeds of fourVitisspecies (V. ripariaMichx.,V. californicaBenth.,V. amurensisRupr., andV. viniferaL.). 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 (Escherichia coli,Salmonella entericaser. Typhimurium, andEnterobacter aerogenes) and Gram-positive (Enterococcus faecalisandStaphylococcus aureus) 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 ofV. amurensisseed extract. Phenolic acids prevailed in the extract fromV. viniferaseeds. TheV. ripariaandV. californicaseed extracts had higher contents of most individual phenolics compared to the otherVitisspecies. 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 againstE. coli. The principal component analysis (PCA) showed discrimination betweenV. vinifera,V. amurensis, and clusteredV. ripariaandV. californicawith respect to variables. To recapitulate, this research demonstrates that seeds of differentVitisspecies, especiallyV. ripariaandV. californica, 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.