Objectives: Methicillin-resistantStaphylococcus aureus (MRSA) remains a major human pathogen. MRSA decolonisation strategies frequently combine chlorhexidine baths and mupirocin nasal ointment. Although MRSA remains widespread in Portuguese hospitals, information regarding resistance to biocides and mupirocin is scarce. We evaluated the prevalence of biocide resistance genes and chlorhexidine and mupirocin non-susceptibility in a representative and well-characterised collection of MRSA isolated in Portuguese hospitals during a 31-year period (1985–2016). Methods: Prevalence of five biocide resistance genes (lmrS, mepA, sepA, qacAB and smr) was determined by PCR. Antibiotic susceptibility was assessed by disk diffusion and by MIC determination using broth microdilution (chlorhexidine) and Etest (mupirocin). Results: Chromosomal genessepA and mepA were detected in all isolates, while lmrS was found in 87.1%. The prevalence of plasmid-borne genes was significant for qacAB (22.4%), associated with the Iberian (ST247-I/IA) clone (P < 0.0001), and low for smr (1.0%) detected among isolates belonging to the ST239-III/IIIvariant clone. Chlorhexidine non-susceptibility (MIC ≥ 4 mg/L) was observed in two isolates belonging to the EMRSA-15 clone (ST22-IV). Non-susceptibility to mupirocin (MIC > 1 mg/L) was significant (15.4%; n = 31) and mainly found among isolates of the EMRSA-15 clone (P < 0.0001; n = 29). One isolate presented low-level mupirocin resistance (MIC = 32 mg/L), and two missense mutations N213D (A637G) and V588F (G1762T) were identified in the ileS gene. Conclusion: Concerningly, we detected a high prevalence of biocide resistance genes and an association of mupirocin and chlorhexidine non-susceptibility with the dominant EMRSA-15 clone in Portuguese hospitals.