Endometritis constitutes a major problem in the management of broodmares; hence, diagnostic tests with a high sensitivity and specificity are highly appreciated. The aim of this study was to compare the results from endometrial, cytologic, and bacteriologic examinations obtained by a newly developed, double-guarded, flushing technique versus standard diagnostic tests, the double-guarded swab and biopsy. The described double-guarded flush technique requires the use of a disposable uterine flushing tube, a sanitary sleeve, a sterile steel speculum, and a 250 mL fluid bag. Endometrial biopsies, swabs, and low-volume lavage samples were obtained from 34 research mares at six different time points in four estrous cycles and were evaluated cytologically and bacteriologically. Endometrial biopsies from the first cycle (n = 34) were examined for the presence of polymorphonuclear neutrophils (PMNs) in the stratum compactum and stratum spongiosum and used as a gold standard for calculation of diagnostic sensitivity and specificity. In all samples, Escherichia coli was most frequently isolated (lavage, 30%; swab, 21%; and biopsy, 12%) followed by β-hemolytic streptococci (lavage, 11%; swab, 8%; and biopsy, 7%). Positive cytology was less likely to occur when E coli was isolated from the diagnostic tests compared with the growth of β-hemolytic streptococci. Isolation of pathogens from uterine samples was highly associated with the presence of PMNs in the stratum compactum and straum spongiosum on histology. Using the presence of PMNs in the tissue specimens as the gold standard for diagnosing endometritis, the sensitivity of low-volume lavage culture was 0.75 and the specificity was 0.72. In conclusion, the double-guarded, low-volume, lavage technique was a rapid and accurate method for diagnosing mares with endometritis, and the risk of false-positive samples is considered to be minimal compared with other flushing techniques described.