BackgroundColonization of the oropharynx with gram-negative bacilli (GNB) is considered a negative prognostic factor in immunocompromised individuals. Hemato-oncologic patients represent a high-risk group due to their immunodeficiencies and associated treatments. This study aimed to determine the rates of oral colonization by GNB, associated factors, and clinical outcomes in patients with hematologic malignancies and solid tumors compared with healthy subjects.MethodsWe conducted a comparative study of hemato-oncologic patients and healthy subjects from August to October 2022. Swabs were taken from the oral cavity; specimens with GNB were identified and tested for antimicrobial susceptibility.ResultsWe included 206 participants (103 hemato-oncologic patients and 103 healthy subjects). Hemato-oncologic patients had higher rates of oral colonization by GNB (34% vs. 17%, P = 0.007) and GNB resistant to third-generation cephalosporins (11.6% vs. 0%, P < 0.001) compared to healthy subjects.Klebsiellaspp. was the predominant genus in both groups. The factor associated with oral colonization by GNB was a Charlson index ≥ 3, while ≥ 3 dental visits per year were a protective factor. Regarding colonization by resistant GNB in oncology patients, antibiotic therapy and a Charlson index ≥ 5 were identified as associated factors, while better physical functionality (ECOG ≤ 2) was associated with less colonization. Hemato-oncologic patients colonized with GNB had more 30-day infectious complications (30.5% vs. 2.9%, P = 0.0001) than non-colonized patients.ConclusionOral colonization by GNB and resistant GNB are prevalent in cancer patients, especially those with higher scores on the severity scales. Infectious complications occurred more frequently in colonized patients. There is a knowledge gap about dental hygiene practices in hemato-oncologic patients colonized by GNB. Our results suggest that patients’ hygienic-dietary habits, especially frequent dental visits, are a protective factor against colonization.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12903-023-03172-y.