Twenty‐five strains, isolated from raw, non‐pasteurised, organic whey samples, were identified phenotypically and genotypically. Biochemical tests were performed, and enzyme profiles, antibiotic resistance and antimicrobial properties were investigated. Sixteen strains were identified as genus Lactobacillus. Based on 16S rDNA gene sequence, the strains were identified as Lb. plantarum and Lb. fermentum. All of the strains had β‐galactosidase activity, and some of them reduced nitrate content. All strains utilised carbohydrates. The tested strains were characterised by low or average lipolytic and esterolytic activity. Moreover, the strains showed low proteolytic activity which is advantageous for their use as starter cultures for foods with low protein content. Strains Lb. fermentum S20, SM1, SM3, S2R and Lb. plantarum SM5 produced harmful N‐acetyl‐β‐glucosaminidase; moreover, the strain S20 produced also β‐glucuronidase. None of the strains produced α‐chymotrypsin. In phenotypic studies, most of the test strains were susceptible to gentamicin, ampicillin, tetracycline, chloramphenicol, penicillin and erythromycin. Strains Lb. plantarum S1 and Lb. fermentum S4, S7, S8, S10, SM1 and SM3 did not possess any transfer resistance genes. Antagonistic activity of the culture LAB strains was assessed as high or moderate in relation to the indicator strains, with the greatest zones of inhibition for E.coli and the smallest for L. monocytogenes ATCC 15313. This study reveals that the LAB strains isolated from organic whey have high potential for food application. Some strains of species Lb. fermentum (S4, S7, S8, S10) have been identified as the best candidates.