Starter cultures composed of lactic acid bacteria (LAB) were developed based on the genotypic and phenotypic characterisation of isolates belonging to dominant groups of bacteria in spontaneous rye wholemeal sourdoughs. Combinations of strains have been evaluated on an industrial scale in the sourdough fermentation process. Wholemeal rye bread was prepared using sourdoughs obtained with 3 new starter cultures, and compared to bread made using the commercial culture (LV2). All newly developed cultures used for the preparation of wholemeal rye bread allowed to obtain better quality products as compared to the LV2 based bread. The best results were obtained when the culture containing Lactiplantibacillus plantarum 2MI8 and exopolysaccharide (EPS)-producing Weissella confusa/cibaria 6PI3 strains was applied. The addition of yeast during sourdough breads production, especially the one prepared from mentioned above starter culture, significantly improved their organoleptic properties, their volume and crumb moisture was increased, and also the crumb acidity and hardness was reduced. Fermentation of rye wholemeal dough, especially without the yeast addition, resulted in a significant reduction in the content of higher inositol phosphates as compared to the applied flour, which is associated with improved bioavailability of minerals. The results of this study prove that the investigated new starter cultures can be successfully applied in wholemeal rye bread production.