•The most dominant polyphenol in blackthorn is neochlorogenic acid.•Blackthorn extracts inhibit α-amylase and α-glucosidase activity.•Antiproliferative effect on HT29 varies among blackthorn samples.•Quercetin glycosides and anthocyanins are the major contributors of the bioactive response.•PCA analysis is a useful tool to distinguish blackthorn genotypes according to chemical analysis. The aim of this work was to investigate and compare the phenolic profile of 15 wild growing blackthorn (Prunus spinosa L.) genotypes from the slopes of Fruška Gora mountain in north Serbia. Their effect in inhibiting i) α-amylase and α-glucosidase activities and ii) colorectal cancer cell line (HT29) growth was also studied. Blackthorn fruit extracts exhibited high phenolic content being enrich in anthocyanins. Principal component analysis was used to correlate the bioactive response with phenolic composition. It was found that derivatives quercetin and anthocyanin peonidin are the major contributors of the inhibition of carbohydrates hydrolyzing enzymes as well as with the antiproliferative effect of blackthorn. Among all samples, the genotype from Beška locality showed the higher capacity in inhibiting alpha-amylase, alpha-glucosidase and HT29 cell growth. Because of high anthocyanin content and higher bioactive response, these genotypes could be recommended for the further cultivation and investigation.