Neomycin is a concentration-dependant aminoglycoside antimicrobial used to treat enterotoxigenic Escherichia coli (ETEC)-related post-weaning diarrhoea (PWD) in pigs. The objective was to compare the efficacy of neomycin administered in a single high dose (50,000 IU/kg) and a standard dose and frequency (25,000 IU/kg daily for 3 consecutive days) in reducing the number of pigs with clinical PWD. We also aimed to evaluate the development of antimicrobial resistance in E. coli following neomycin treatment. The study was performed in a Danish herd not using medicinal zinc oxide and experiencing outbreaks of PWD caused by ETEC in the first week after weaning. Pigs from six batches with perianal faecal staining on days 4–6 after weaning and a faecal score of 3–4 were ear tagged and treated with neomycin. Pens were randomly assigned to a treatment group before inclusion. A total of 772 pigs (471 in the control group and 301 in the experimental group) were included and treated orally. The apparent prevalence of diarrhoea on the first day of inclusion across six batches (n = 1,875) was 27%. The efficacy of the neomycin treatment strategy was 86% for the control group and 91% for the single high-dose group (p = 0.043), and the mean percentage (standard deviation (sd)) of haemolytic E. coli-like colonies was 12% (26) and 26% (37) (p < 0.001), respectively. Neomycin resistance did not differ between groups. Before treatment, all analysed isolates were identified as ETEC (n = 142), while after treatment, 91% were identified as ETEC (n = 69) and 9% (n = 7) as non-ETEC E. coli (without fimbria or toxins). A higher cure rate in the single high-dose group suggests that ETEC-related PWD can be treated with a single high dose of 50,000 IU/kg of neomycin, thereby reducing antimicrobial use by 33% compared to the standard treatment of 25,000 IU/kg for 3 consecutive days. The study indicated a higher number of haemolytic E. coli in the single high-dose group after treatment, but no evidence of increased neomycin resistance in coliforms was observed compared to the standard treatment.