Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were: bacterial culturing of faecal samples from three pigs (per pen) with clinical diarrhoea and subsequent testing for virulence genes in E. coli isolates; bacterial culturing of pen floor samples and subsequent testing for virulence genes in E. coli isolates; qPCR testing of pen floor samples in order to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes for one or more adhesin factors and one or more enterotoxins were detected. Results: A total of 208 E. coli colonies from pig samples and 172 E. coli colonies from pen floor samples were isolated. Haemolytic activity was detected on blood agar plates in 111 (29.2%) of the 380 colonies that were isolated. The only adhesin factor detected in this study was F18. When comparing bacterial culture or qPCR testing of pen floor samples with detection of ETEC-positive diarrhoeic pigs by culture, agreement was found in 26 (83.9%, Kappa = 0.665) and 23 (74.2%, Kappa = 0.488) of the pens, respectively. Agreement was observed between the detection of ETEC by bacterial culture and qPCR in the same pen floor sample in 26 (83.9%, Kappa = 0.679) pens. Conclusion: We observed an acceptable agreement for the detection of ETEC-positive diarrhoeic nursery pigs in pen samples for both bacterial culture of pen floor samples and qPCR. This study showed that both bacterial culture and qPCR testing of pen floor samples can be used as a diagnostic approach for detecting groups of ETEC-positive diarrhoeic nursery pigs.