The primary aim of this experiment was to investigate the bioactivity potential and polyphenolic profile of defatted raspberry seeds (DRS) extracts from three varieties (Willamette, Meeker, and Polka) using the in vitro tests HPLC-DAD and UHPLC-Triple-TOF-MS. Extracts were obtained using ultrasound-assisted extraction (UAE) or hydrolysis. The antioxidant activity of the extracts was tested using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic) cation (ABTS), and ferric reducing antioxidant power (FRAP) assays. Furthermore, the extracts were tested for antimicrobial activity using the disk diffusion method for four bacterial cultures (Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Salmonella enterica subsp. enterica Enteritidis). In vitro antiproliferative activity was tested using cervical carcinoma (HeLa), breast adenocarcinoma (MCF7), and fetal lung (MRC-5) human cell lines. In total, 32 phenolic compounds were detected in DRS extracts. A small quantity of ellagic acid (EA) was in free form, while EA content increased after the hydrolysis process. The extracts from the Meeker variety exhibited the highest antioxidant activity, analyzed with DPPH and FRAP assays, while extracts from the Polka variety had the highest activity towards ABTS+ radical scavenging activity. The UAE samples expressed higher antiproliferative activity in comparison to hydrolysis extracts. The results indicate that DRS extracts have certain bioactivity, and their use in the food, cosmetic, and pharmaceutical industries is recommended.