Multidrug resistant Gram-Negative Bacterial Infections (MR-GNBI) are an increasing cause of mortality in acute myeloid leukemia (AML), compromising the success of antineoplastic therapy. We prospectively explored a novel strategy, including mandatory fluoroquinolone prophylaxis, weekly surveillance cultures (SC) and targeted antimicrobial therapy for febrile neutropenia, aimed to reduce infectious mortality due to MR-GNBI. Over 146 cycles of chemotherapy, cumulative incidence of colonization was 50%. Half of the colonizations occurred in the consolidation phase of treatment. Application of this strategy led to a significant reduction in the incidence of GNB and carbapenemase-producing Klebisella pneumoniae (cpKp) species, resulting in a reduction of infectious mortality (HR 0.35 [95%, CI 0.13–0.96], p = 0.042). In multivariate analysis, fluroquinolone prophylaxis in addition to SC was associated with improved survival (OR 0.55 [95% CI 0.38–0.79], p = 0.001). Targeted therapy for colonized patients did not overcome the risk of death once cpKp or XDR Pseudomonas aeruginosa infections were developed. Mortality rate after transplant was similar between colonized and not colonized patients. However only 9% of transplanted patients were colonized by cpkp. In conclusion, colonization is a common phenomenon, not limited to the induction phase. This strategy reduces infectious mortality by lowering the global incidence of GN infections and the spread of resistant species.