The growth rate of bovine lactic acid bacteria (LAB) in five different culture conditions, and their inhibitory activity against Escherichia coli O157 and F5 in two assays was assessed to identify LAB for potential prophylactic use in cattle. 106 bovine-derived faecal/intestinal LAB were tested in vitro for tolerance to pH 2.0, pH 4.0, 0.15% and 0.3% bile, aerobic incubation, and for inhibitory activity against E. coli O157 (n = 3) and F5 (n = 1). While no LAB grew at pH 2.0, LAB survivability varied between 35% and 100% on the other tests. Exactly 7.6% (8/106) of LAB supernatants inhibited the growth of E. coli in two assays, whereas 6.6% (7/106) of isolates enhanced the growth of all E. coli strains. Partial 16s rRNA gene sequencing of six best isolates (95th percentile) revealed that five were Lactobacillus plantarum and one Pediococcus acidilactici. Lactobacillus plantarum with acid/bile and aerobic resistance and inhibitory activity against E. coli O157 and F5 inhabit the intestinal tract of healthy cattle. Some LAB may enhance E. coli growth. Lactobacillus plantarum and P. acidilactici are natural plant micro-organisms and studied silage inoculants. Their identification from gastrointestinal samples of healthy cattle is prophylactically promising.