Introduction: Synthetic colourants are subject to various allergies. As a result, the use of plant-based additives has proved promising. Objective: It is in this perspective that our objective was undertaken to investigate natural colourants from edible cactus. Opuntia species populations were singlesequence repeat (SSR) fingerprinted, the analysis of betalains and phenolic compounds and the evaluation of their biological potentials were also characterised. Results: Photometric quantification of betalains and phenols showed an interspecific variation across Opuntia species. Opuntia ficus indica fruits showed the highest betalain [betaxanthins; 843.67 and betacyanins; 1400 mg/100 g dry weight (DW)] and polyphenol contents. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis showed that the variation of individual phenols profile was influenced by interspecific and genetic factors. Isorhamnetin-O-(di-deoxyhexosylhexoside) was the major compound and its content varied according to Opuntia species, while catechol was the predominant phenolic compound in O. humifusa with 1.88 μg/g DW. Concerning cactus species, Opuntia colourants exhibited a potent antiradical activity [half maximal inhibitory concentration (IC 50) up to 1 μg/mL]. Opuntia species were effective against Gram-positive and Gram-negative bacterial strains [inhibition zone (IZ) up to 27 mm]. A high genetic diversity within Opuntia genotypes based on SSR markers was revealed. UPGMA (unweighted pair group method with arithmetic mean) dendrogram and PCoA (principal coordinate analysis) based on natural pigments and antimicrobial profiles indicated significant variation. The correlation approach proved the presence of a probably metabolic relationship between genetic markers, pigments and their biological activities. Conclusion: A possible association between molecular approach and metabolic profile analysis of Opuntia allows tracing the relationship among species for its genetic conservation.