Abstract Ninety-eight Lactobacillus (Lb.) strains were screened to select those with the best antimicrobial and probiotic properties. Firstly, a screening based on growth kinetics under gastrointestinal tract (GIT) conditions allowed to select 28 strains, which were assayed for autoaggregation, coaggregation and hydrophobicity. 7.1% were strongly autoaggregating strains after 4 h of incubation. Strains belonging to Lb. plantarum and Lb. paraplantarum species showed the highest coaggregation percentages with the pathogens tested (Salmonella choleraesuis and Staphylococcus aureus). Regarding hydrophobicity, there was correlation between the results obtained for xylene and toluene. Principal Component Analysis (PCA) of these results allowed to select 20 strains as potential probiotics, that were used for further assays of biofilms formation (ranged between 53 and 75%) and adhesion to Caco-2/TC7 cells (around 51–97%). Furthermore, 51% of the ninety-eight strains showed antimicrobial activity against four species indicators (Salmonella choleraesuis, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes). Results from all the assays performed allowed to select 12 strains attending to either their antimicrobial activity or the probiotic potential and only two of them Lb. paracasei Lb38 and Lb. brevis Lb99, stood up for both properties. Therefore, these strains could be proposed to be used as biocontrol agents in the food industry.