Probiotics are generally known as live, non-pathogenic bacteria that provide health benefits when consumed. Milk and other fermented dairy products serve as the most common sources of probiotics. This study was carried out to assess the antagonistic activity of probiotic Lactobacillus species isolated from a locally fermented milk sample (Nono) against clinical isolates of Escherichia coli from diarrhoeic stools of children. The milk sample was purchased from a local vendor within Samaru, Zaria. A portion of the milk was immediately taken to the Laboratory of the National Animal Production Research Institute (NAPRI), Ahmadu Bello University (ABU), Zaria, for proximate analysis. Another portion was also taken to the Laboratory of the Department of Microbiology, ABU, Zaria, where it was serially diluted, inoculated onto De Man Rogosa and Sharpe (MRS) Agar using the spread plate method and incubated anaerobically at 37^oC for 48 hrs. Isolates with characteristic colonial morphology of Lactobacillus species were further identified using Gram staining and biochemical tests, followed by screening for probiotic properties. These were then tested against the clinical isolates of Escherichia coli using the agar well diffusion technique. Results for the proximate composition of the milk revealed that it contains moisture (84.57%), crude protein (4.85 %), crude fibre (0.00%), fat (2.25%), ash (1.15%) and carbohydrates (7.18%). Biochemical tests also confirmed the isolates to be Lactobacillus acidophilus and Lactobacillus fermentum. Out of 18 clinical isolates of Escherichia coli tested with the probiotic bacteria, 7 (38.8%) were susceptible, producing a zone of inhibition ranging between 09 and 21mm. From this study, it was concluded that Lactobacillus species (which are probiotic bacteria) have antagonistic activity against pathogens such as E. coli. It can therefore be recommended that children with diarrhoea be treated with foods such as fermented dairy products containing these lactic acid bacteria.