•The phenolic composition of 25 varieties of roselle (Hs) was determined.•A total of 7 new compounds were tentatively characterized in Hs extracts.•The Hs extracts exhibited potent antioxidant and antimicrobial activities.•Roselle varieties were classified based on their anthocyanin content. This study reports the phytochemical profile (phenolics, flavonoids and anthocyanins), the antioxidant capacity and the antibacterial activity of the ethanolic extracts of a collection of 25 Mexican Hibiscus sabdariffa (Hs) varieties with different calyx color intensities, from green–yellow to deep red, cultivated in the same condition. A great variety of phenolic compounds were identified in the different extracts using HPLC-DAD-ESI-TOF-MS, mainly phenolic acids, flavonoids, and anthocyanidin, some of which have been described for the first time. The total phenolic, flavonoid and anthocyanidin contents showed great variation in the different Hs extracts, ranging from 2400±300 to 10,000±400mg gallic acid equivalents (GAE)/100g dry calyx (dc), from 419±2 to 2260±70mg quercetin/100g dc, and from 0 to 4408mg/100g dc with different ratios of delphinidin:cyanidin, respectively. Furthermore, the antioxidant capacity determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay presented the same behavior, with values that varied from 27.4±0.3 to 112±8mol equivalent trolox/g dc. In addition, the antibacterial activity of the HS extracts was assayed against Gram-negative (Escherichia coli, Salmonella enteritidis) and Gram-positive (Staphylococcus aureus, Micrococcus luteus) microorganisms, demonstrating that the ethanol extracts were effective against all the bacterial strains tested, showing a greater effect against Gram-positive bacteria. Finally, a multivariate analysis for the classification of the Hs varieties has been carried out based on the anthocyanidin content. The results reported in this paper shows for the first time antibacterial and phytochemistry diversity of the different varieties of Hs, which highlights the importance of a correct description of Hs materials used in research papers.