The aim of this work was to identify and select new probiotic strains among majority intestinal bifidobacterial species from healthy Spaniards. One hundred and eighty isolates belonging to seven Bifidobacterium species were subjected to a subtractive system of in vitro analysis addressing beneficial and undesirable traits. Approx. 45% of the isolates were able to grow in 2% bovine bile, and about 20% of these grew at pH 3.5. Undesirable enzymatic activities, such as alpha-chymotrypsin, beta-glucuronidase and N-acetyl-beta-glucosaminidase were not detected. Atypical antibiotic resistances were not observed, except for tetracycline resistance in a single strain. Intestinal pathogens were inhibited to some extent by all analysed strains. All selected strains adhered to human epithelial cells in a strain-dependent manner, and none was able to degrade pig mucin. Based on these in vitro analyses, strains of Bifidobacterium catenulatum, Bifidobacterium longum and Bifidobacterium pseudocatenulatum are here proposed as new probiotic candidates. Although in vivo analyses are still needed, these strains belonging to unusual species in the portfolio of probiotic suppliers are thought to be more appropriated than those currently in use, as they show desirable properties and are preponderant among human intestinal populations.