The present study was aimed to investigate the polyphenolic profile of a pepper (Capsicum annuum L.) extract from Algeria and evaluate its biological activity. The total polyphenol content of the extract was determined as 1.373 mg of gallic acid equivalents (±0.0046), whereas the flavonoids were determined as 0.098 mg of quercetin (±0.0015). The determination of the complete polyphenolic profile of the extract was achieved by liquid chromatography with an RP‐amide column in combination with photodiode array and mass spectrometry detection through an electrospray ionization interface. A total of 18 compounds were identified, of which five were reported for the first time in the sample tested. Quercetin rhamnoside was the most abundant compound (82.6 μg/g of fresh pepper) followed by quercetin glucoside (19.86 μg/g). The antioxidant activity and antimicrobial effects were also determined. For the antimicrobial tests assessed against Gram‐positive and Gram‐negative bacteria, kaempferol showed the strongest inhibitory effect followed by quercetin and caffeic acids. In the study of the cytotoxicity of the extract, the cancer cells (U937) were more affected than the normal cells (peripheral blood mononucleated cells), with more than 62% inhibition at the highest concentration.