Objectives: To evaluate the impact of different antibiotic strategies on acquisition of resistant microorganisms. Methods: A prospective study was conducted over a 44 month period in a single ICU. Four empirical antibiotic strategies for ventilator-associated pneumonia (VAP) were sequentially implemented. Over the initial 10 months, patient-specific antibiotic therapy was prescribed; then, 4 month periods of prioritization or restriction rotation cycles of various antimicrobial agents were implemented for a total of 24 months; and, finally, during the last 10 months (mixing period) the first-line antibiotic for VAP was changed following a pre-established schedule to ensure maximum heterogeneity. Antibiotic consumption was closely monitored every month, and antimicrobial resistance patterns were regularly assessed. Antimicrobial heterogeneity was estimated using a modified Peterson index (AHI) measuring the ratios for the five most used antibiotics. Colonization by targeted microorganisms and susceptibility patterns were compared with the patient-specific period. Results: Higher diversity of antibiotic prescription was obtained during patient-specific therapy (AHI = 0.93) or mixing periods (AHI = 0.95) than during prioritization (AHI = 0.70) or restriction periods (AHI = 0.68). High homogeneity was associated with increases in carbapenem-resistant Acinetobacter baumannii (CR-Ab) [relative risk (RR) 15.5; 95%CI 5.5–42.8], extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae (RR 4.2; 95%CI 1.9–9.3) and Enterococcus faecalis (RR 1.7; 95%CI 1.1–2.9). During the restriction period, incidence of ESBL-producing Enterobacteriaceae and E. faecalis returned to patient-specific rates but CR-Ab remained higher. Conclusions: Antibiotic prescription patterns balancing the use of different antimicrobials should be promoted to reduce the selection pressure that aids the development of resistance.