Previous studies of direct-fed microbial (DFM) supplements showed variable effects on the microbiota and physiology of dairy cows. The main aims of this study were to investigate the milk microbiota of cows supplemented with a lactobacilli-based DFM compared to untreated cows; describe the changes; and quantify the association between the taxa and cow productivity. The study followed seventy-five Holstein–Friesian dairy cows supplemented with a DFM over 16 months compared to seventy-five non-supplemented cows. Twenty-five cows from each group were sampled for microbiota analysis. The top taxa significantly associated with the variables were as follows: Age (Mammaliicoccus_319276, Turicibacter), milk production (Turicibacter, Bifidobacterium_388775), DIM (Stenotrophomonas_A_615274, Pedobacter_887417), milk fat percentage (Pseudomonas_E_647464, Lactobacillus), calendar month (Jeotgalicoccus_A_310962, Planococcus), milk protein percentage (Tistrella, Pseudomonas_E_650325), experimental group (Enterococcus_B, Aeromonas), SCC (Paenochrobactrum, Pseudochrobactrum), and trimester of pregnancy (Dyadobacter_906144, VFJN01 (Acidimicrobiales)). These were identified using multivariable analysis. Twenty-six genera were associated with the differences between experimental groups, including Pseudomonas, Lactococcus and Staphylococcus. Microbial taxa that changed in relative abundance over time included Atopostipes, Brevibacterium and Succinivibrio. Many of these genera were also part of the core microbiota. Supplementation with the DFM significantly altered the milk microbiota composition in the dairy cows, highlighting the impact of long-term DFM supplementation on microbial communities.