Diarrhoeal diseases remain a major public health problem and are among the leading causes of morbidity and mortality in children, particularly in developing countries. Among the bacterial pathogens associated with childhood diarrhoea, Escherichia coli is frequently implicated, with diarrhoeagenic strains contributing significantly to gastrointestinal infections. Although the infection can be treated with antibiotics however the emergence of multi antibiotic-resistant E. coli has complicated treatment therefore the need for alternative antibacterial therapies. This study therefore evaluates cheese whey for antibacterial activity against diarrhoeagenic E. coli isolated from stool samples of diarrhoeic children in the bid of searching for alternative therapies for treating bacterial diarrhoea caused by E. coli. The influence of types of coagulants in whey production on its antibacterial properties was also studied. A total of 55 stool samples was collected from diarrhoeal children at selected primary health centers and government hospitals in Owo, Ondo State, Nigeria. The collected stools were analyzed using standard microbiological, biochemical and molecular techniques to identify the types of bacteria present. The antibiogram profile of the isolated E. coli was determined using standard microbiological assays. The antibacterial activity of commercially prepared whey (CPW) was compared with that of the laboratory prepared whey (LPW) against the isolated E. coli using agar well diffusion assay. So also the effect of types of coagulants used in the production of whey on its antibacterial activity using standard methods. E. coli was isolated in 47.3% of the stool samples examined. Other bacterial species isolated are Staphylococcus aureus (21.8%), Pseudomonas aeruginosa (16.4%), and Salmonella enterica (14.6%). The antibiogram profile of the isolated E. coli revealed high levels of multi-antibiotic resistance (MAR) index (0.4-1.0) among the isolates to pefloxacin, amoxicillin, augmentin and streptomycin but were susceptible to ciprofloxacin. CPW demonstrated antibacterial activity against some of the isolated E. coli whereas LPW exhibited broader and stronger antibacterial activity on all the test E. coli isolates except one. The inhibition mediated by LPW was superior to that mediated by ciprofloxacin on 7 (26.9%) of the E. coli isolates and was at per with the inhibition mediated by ciprofloxacin on most of the E. coli isolates. Whey prepared using Calotropis procera as coagulant showed the highest growth inhibitory activity compared with whey prepared using lime and alum. The findings demonstrate that cheese whey possesses antibacterial properties against the isolated diarrhoeagenic E. coli, with efficacy influenced by coagulant type. Therefore, cheese whey can be explored in the development of a more effective antibacterial drug that can be exploited in the management of diarrhoea caused by E. coli especially in the absence of ciprofloxacin which is the most effective antibiotic against the isolated diarrhoeagenic E. coli.