This research effort aimed at isolating and phenotypically characterizing lactic acid bacteria (LAB) isolates from a spontaneous rye sourdough manufactured following traditional protocols, as well as at evaluating their antimicrobial and antifungal properties as key features for future industrial applications. Thirteen LAB strains of potential industrial interest were isolated and identified to species-level via PCR. Most of the sourdough isolates showed versatile carbohydrate metabolisms. The Leuconostoc mesenteroides No. 242 and Lactobacillus brevis No. 173 demonstrated to be gas producers; thus, revealing their heterofermenter or facultative homofermenter features. Viable counts higher than 7.0 log₁₀ (CFU/mL) were observed for Lactobacillus paracasei No. 244, Lactobacillus casei No. 210, L. brevis No. 173, Lactobacillus farraginis No. 206, Pediococcus pentosaceus No. 183, Lactobacillus uvarum No. 245 and Lactobacillus plantarum No. 135 strains, after exposure at pH 2.5 for 2 h. Moreover, L. plantarum No. 122, L. casei No. 210, Lactobacillus curvatus No. 51, L. paracasei No. 244, and L. coryniformins No. 71 showed growth inhibition properties against all the tested fifteen pathogenic strains. Finally, all LAB isolates showed antifungal activities against Aspergillus nidulans, Penicillium funiculosum, and Fusarium poae. These results unveiled the exceptionality of spontaneous sourdough as a source of LAB with effective potential to be considered in the design of novel commercial microbial single/mixed starter cultures, intended for application in a wide range of agri-food industries, where the antimicrobial and antifungal properties are often sought and necessary. In addition, metabolites therefrom may also be considered as important functional and bioactive compounds with high potential to be employed in food and feed, as well as cosmetic and pharmaceutical applications.