<i>Clostridioides difficile</i> is a leading cause of healthcare-associated infections worldwide. Currently, there is a lack of consensus for an optimal diagnostic method for <i>C. difficile</i> infection (CDI). Multi-step diagnostic algorithms use enzyme immunosorbent analysis (EIA)-based detection of <i>C. difficile</i> toxins TcdA/TcdB in stool, premised on the rationale that EIA toxin-negative (Tox-) patients have less severe disease and shorter diarrhea duration. The aim of this study was to characterize toxigenic (i.e. <i>tcdA/tcdB</i>-positive) <i>C. difficile</i> strains isolated from diarrheic patient stool with an EIA Tox- (i.e. “discrepant”) CDI diagnostic test result. Recovered strains were DNA fingerprinted (ribotyped), subjected to multiple toxin, genome and proteome evaluations, and assessed for virulence. Overall, of 1243 <i>C. difficile</i>-positive patient stool specimens from Southern Arizona hospitals, 31% were discrepant. For RT027 (the most prevalent ribotype)-containing specimens, 34% were discrepant; the corresponding RT027 isolates were cytotoxic to cultured fibroblasts, but their total toxin levels were comparable to, or lower than, the historic low-toxin-producing <i>C. difficile</i> strain CD630. Nevertheless, these low-toxin RT027 strains (LT-027) exhibited similar lethality to a clade-matched high-toxin RT027 strain in Golden Syrian hamsters, and heightened colonization and persistence in mice. Genomics and proteomics analyses of LT-027 strains identified unique genes and altered protein abundances, respectively, relative to high-toxin RT027 strains. Collectively, our data highlight the robust virulence of LT-027 <i>C. difficile</i>, provide a strong argument for reconsidering the clinical significance of a Tox- EIA result, and underscore the potential limitations of current diagnostic protocols.