Background: Contamination of the hospital environment with pathogens carrying antimicrobial resistance (AMR) genes may contribute to transmission. Inadequate environmental cleaning and poor hand hygiene compliance may transfer AMR pathogens resulting in colonization and/or infection. Methods and materials: A cross-sectional study was conducted in the obstetric and neonatal wards at Tygerberg Hospital in Cape Town, South Africa between February and December 2016. To establish the density of hospital environmental contamination, we sampled 378 high- and low-touch surfaces for the presence of the following resistance genes: CTX-M15, NDM, OXA-48 and KPC. We screened 180 peripartum women and 92 hospitalized neonates to establish the prevalence of patients’ rectal colonization with bacteria carrying AMR genes. The adequacy of environmental cleaning and hand hygiene compliance rates in the neonatal and obstetric wards was assessed using a validated Infection Control Assessment Tool. Univariate analysis models were built to identify associations between environmental contamination and patients’ colonization. Results: Overall prevalence of environmental contamination with AMR genes was 5.8% (22/378) including 19 (86.4%) samples with CTX-M15 and 3 (13.6%) with OXA-48. Low-touch surfaces had significantly higher levels of contamination with CTX-M15 than high-touch surfaces (8/72 [11.1%] versus 11/306 [3.6%]; p = 0.002). The environmental cleaning score was similar in neonatal (80%) and obstetric (81%) wards; the mean hand hygiene compliance rates were 63% and 50% in neonatal and obstetric wards respectively. Five percent (14/272 patients) had CTX-M15 rectal colonization, with higher rates among neonates than peripartum women (9/92 [9.8%] vs. 5/180 [2.8%]; p = 0.013). CTX-M15 hospital environment contamination was not a predictor of patients’ CTX-M15 colonization status [positive swab versus negative OR = 1.8 (95% CI 0.2–22), p = 0.6. Conclusion: Levels of hospital environmental contamination with AMR genes were low (5.8%), and did not support a link between hospital contamination and maternal colonization with CTX-M15.