Healthcare-acquired infections (HAIs) contribute to maternal and neonatal morbidity and mortality, especially in low- and middle-income countries (LMICs). Deficient environmental health (EH) conditions and infection prevention and control (IPC) practices in healthcare facilities (HCFs) contribute to the spread of HAIs, but microbial sampling of sources of contamination is rarely conducted nor reported in low-resource settings. The purpose of this study was to assess EH conditions and IPC practices in Malawian HCFs and evaluate how EH deficiencies contribute to pathogen exposures and HAIs, and to provide recommendations to inform improvements in EH conditions using a mixed-methods approach. Thirty-one maternity wards in government-run HCFs were surveyed in the three regions of Malawi. Questionnaires were administered in parallel with structured observations of EH conditions and IPC practices and microbial testing of water sources and facility surfaces. Results indicated significant associations between IPC practices and microbial contamination. Facilities where separate wards were not available for mothers and newborns with infections and where linens were not used for patients during healthcare services were more likely to have delivery tables with surface contamination (relative risk = 2.23; 1.49, 3.34). E. coli was detected in water samples from seven (23%) HCFs. Our results suggest that Malawian maternity wards could reduce microbial contamination, and potentially reduce the occurrence of HAIs, by improving EH conditions and IPC practices. HCF staff can use the simple, low-cost EH monitoring methods used in this study to incorporate microbial monitoring of EH conditions and IPC practices in HCFs in low-resource settings.