Methicillin-resistant Staphylococcus aureus (MRSA) infection is a leading cause of hospitalization and medical visits among individuals experiencing homelessness and also among persons who inject drugs (PWID), populations with significant overlap in urban centers in the USA. While injection drug use is a risk factor for MRSA skin infections, MRSA is also known to transmit easily in crowded, public locations in which individuals have reduced personal hygiene. Individuals in urban centers who experience homelessness or drug addiction may spend significant amounts of time in environments where MRSA can be easily transmitted, and may also experience reduced access to facilities to maintain personal hygiene. We assessed the relationship between homelessness, personal hygiene, and MRSA nasal colonization, a proxy for MRSA infection risk, in a study of PWID in Boston, MA (n = 78). Sleeping in a homeless shelter for at least one night in the last 3 months was significantly associated with MRSA nasal colonization (OR 3.0; p = 0.02; 95% CI 1.2, 7.6). Sleeping at more than one place during the last week (considered a metric of elevated housing instability) was also associated with a threefold increase in odds of MRSA nasal colonization (OR 3.1; p = 0.01; 95% CI 1.3, 7.6). MRSA nasal colonization was strongly associated with use of public showers (OR 13.7; p = 0.02; 95% CI 1.4, 132.8), although few people in this study (4 of 78) reported using these public facilities. Sharing bedding with other people was also associated with increased risk of MRSA colonization (OR 2.2; p = 0.05; 95% CI 1.0–4.7). No associations between hand hygiene, frequency of bathing or clothes laundering, or street sleeping were observed. Use of public facilities supporting persons experiencing homelessness and housing instability, including shelters and public showers, is associated with an increased risk of MRSA nasal colonization in this study. Personal hygiene behaviors appear less associated with MRSA nasal colonization. Environmental assessments of MRSA contamination in homeless shelters and public sanitation facilities are warranted so as to inform appropriate intervention activities.