Background: In the Erasmus MC University Medical Center, Rotterdam, the Netherlands, patients considered at risk for carrying highly resistant microorganisms (HRMO) are placed in isolation on admission, until tested negative for HRMO (ie, targeted screening). Patients without risk factors are not routinely screened (ie, nontargeted screening). However, nontargeted screening could identify patients colonized with HRMO missed by targeted screening. To determine the additional value of nontargeted screening, we compared the outcomes of the nontargeted screening approach with all available clinical cultures. Objective: We aim to identify patients colonized with HRMO, but missed by targeted screening, and to determine whether non-targeted screening has additional value. Methods: For the MOVE study, nontargeted admission and discharge cultures (nose and perianal) were obtained from randomly selected patients admitted to specific wards, regardless of HRMO risk factors. This study was part of a research initiative to identify the relation of a contaminated environment with the risk of becoming infected or colonized on a patient level. All bacteriological clinical samples positive for at least 1 HRMO from January 1, 2018, until August 31, 2019, were compared with the nontargeted screening samples. Samples were screened for methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) as well as highly resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, and Enterobacteriales. Broth enrichment was used for all cultures. Results: During the study period, 50,653 patients were admitted. 706 patients (1%) had a clinical sample positive for at least 1 HRMO during their hospital stay. 936 (1.8%) patients were included in the nontargeted screening for the MOVE study, and 40 patients were found to have at least 1 culture positive for HRMO (4.3%). Among these 40 patients, 28 were positive at admission and 12 were positive at discharge. Extended-spectrum β-lactamase (ESBL)–producing Enterobacteriales were most prevalent (n = 36, 90.0%) both at admission and discharge (n = 26 and n = 10, respectively). At admission, 1 patient was identified with MRSA and 1 patient was positive for vancomycin-resistant E. faecium (VRE). At discharge, 1 patient was identified with VRE and 1 had Verona Integron-encoded Metallo-β-lactamase (VIM)–positive P. aeruginosa. Conclusions: Our results show that the current targeted screening does not identify all HRMO carriers. Furthermore, patients who acquire an HRMO during admission are missed. The nontargeted screening identified 40 unknown carriers (4.3%). The limitations of the study are the restricted number of sample sites and the fact that we were unable to culture all patients. Therefore, it is likely that our study shows an underestimation of the true number of patients with HRMO.