Abstract Introduction Harboring sensitive strains may prevent acquisition of resistant pathogens by competing for colonization of ecological niches. Competition may be relevant to decolonization strategies that eliminate sensitive strains and may predispose to acquiring resistant strains in high-endemic settings. We evaluated the impact of colonization with methicillin-sensitive Staphylococcus aureus (MSSA) and vancomycin-sensitive enterococci (VSE) on acquisition of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), respectively, when controlling for other risk factors. Methods We conducted a nested case-control study of patients admitted to eight ICUs performing admission and weekly bilateral nares and rectal screening for MRSA and VRE, respectively. Analyses were identical for both pathogens. For MRSA, patients were identified who had a negative nares screen and no prior history of MRSA. We evaluated predictors of MRSA acquisition, defined as a subsequent MRSA-positive clinical or screening culture, compared to those with a subsequent MRSA-negative nares screen within the same hospitalization. Medical records were reviewed for the presence of MSSA on the initial MRSA-negative nares screen, demographic and comorbidity information, medical devices, procedures, antibiotic utilization, and daily exposure to MRSA-positive patients in the same ward. Generalized linear mixed models were used to assess predictors of acquisition. Results In multivariate models, MSSA carriage protected against subsequent MRSA acquisition (OR = 0.52, CI: 0.29, 0.95), even when controlling for other risk factors. MRSA predictors included intubation (OR = 4.65, CI: 1.77, 12.26), fluoroquinolone exposure (OR = 1.91, CI: 1.20, 3.04), and increased time from ICU admission to initial negative swab (OR = 15.59, CI: 8.40, 28.94). In contrast, VSE carriage did not protect against VRE acquisition (OR = 1.37, CI: 0.54, 3.48), whereas hemodialysis (OR = 2.60, CI: 1.19, 5.70), low albumin (OR = 2.07, CI: 1.12, 3.83), fluoroquinolones (OR = 1.90, CI: 1.14, 3.17), third-generation cephalosporins (OR = 1.89, CI: 1.15, 3.10), and increased time from ICU admission to initial negative swab (OR = 15.13, CI: 7.86, 29.14) were predictive. Conclusions MSSA carriage reduced the odds of MRSA acquisition by 50% in ICUs. In contrast, VSE colonization was not protective against VRE acquisition. Studies are needed to evaluate whether decolonization of MSSA ICU carriers increases the risk of acquiring MRSA when discharging patients to high-endemic MRSA healthcare settings. This may be particularly important for populations in whom MRSA infection may be more frequent and severe than MSSA infections, such as ICU patients.