Escherichia coli is an important commensal of our gut, however, many pathogenic strains exist, causing various severe infections in the gut or beyond. Due to several antibiotic resistance patterns of E. coli, research of alternative treatments or adjuvant therapy is important. One of these is the use of probiotics as antagonistic agents against E. coli. Most published studies investigate only one strain of E. coli and single-strain probiotics. The objectives of this study were to evaluate the antagonistic activity of selected single-strain and multi-strain probiotic supplements against selected clinical E. coli pathotypes using the in vitro agar spot test and the co-culturing method. Molecular methods were used to determine the presence of the genus lactobacilli and bifidobacteria as well as certain selected strains in the probiotic supplements. The agar-spot test showed that the multi-strain probiotics were more effective than the single-strain probiotics. On the other hand, the co-culturing method showed the opposite result, indicating that results are importantly influenced by the chosen method. The most effective single-strain probiotics against E. coli strains were Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus reuteri DSM 17938. The most effective multi-strain probiotics contained lactobacilli, bifidobacteria and enterococci strains, thus proving that most effective probiotics against E. coli strains are the lactic acid bacteria and bifidobacteria. The overall results from both in vitro tests reveal that all selected probiotics exhibited an antagonistic activity against all E. coli strains. From a public health perspective probiotics have thus proved to be successful in inhibiting the growth of E. coli and could therefore be used as adjuvant therapy or alternative therapy in E. coli infections.