ABSTRACT Background Technologies and life support management have enhanced the survival of preterm infants. The immune system of newborns is immature, which contributes to the occurrence of healthcare-associated infections. The overlap of several conditions with neonatal sepsis and the difficulty of diagnosis and laboratory confirmation during this period result in a tendency to over-treat neonatal sepsis. The use of antimicrobial agents is a risk factor for multidrug-resistant bacterial infections. This work aimed to perform a systematic review of the relationship between inadequate use of antimicrobial agents and increase in neonatal sepsis related to healthcare assistance, due to bacterial resistance. Methods Our population, exposition, comparison, outcome and study type was as follows: P: hospitalized neonates with sepsis diagnosis, E: inappropriate use of antimicrobial agents, C: adequate use of antimicrobial agents or no indication of infection, O: resistant bacterial infection, and S: original studies. We performed searches in the PubMed, Scopus, Virtual Health Library (Scielo, LILACS, and MEDLINE), and Embase without limits on time, language, and the references of the articles found. Fourteen studies were included and assessed using the Grading of Recommendations, Assessment, Development, and Evaluation, Newcastle, and the Strengthening the Reporting of Observacional Studies in Epidemiology methodologies. Results All studies found were observational and started with a low-quality evidence level in the Grading of Recommendations, Assessment, Development, and Evaluation. Conclusions Despite their low-quality evidence, the studies demonstrated the association between inadequate use of antimicrobial agents and increase of neonatal resistant bacterial healthcare-associated infections in neonatal units. However, there is significant difficulty in conducting high-quality studies in this population due to ethical issues tied to randomized trials. Therefore, new studies should be encouraged to recommend adequate treatment of newborns without increasing the risk of healthcare-associated infections by multidrug-resistant bacteria.