Background: Infection control is a more substantial area of concern in labor and delivery room where nosocomial infection can be acquired in the maternal and child health facility. This study aimed to assess knowledge and practice of midwives in labor room regarding infection control. Design: A descriptive cross-sectional design was conducted. Setting: five main governmental hospitals at Sana'a city. Subjects: 84 midwives who work in delivery rooms were selected. Tools: two tools were used tool I: structured questionnaire consist of two parts, part 1: demographic data and part 2 to assess their knowledge related to universal precaution, tool II: checklist was used to assess the practice. Results: the total knowledge about universal precaution and infection control, slightly more than half of the subjects had good knowledge whereas few had poor knowledge. The majority of them had excellent practice for universal precaution and the there was no statistical significant between total knowledge score and total practice score. Conclusion: The total score of midwife's knowledge related to universal precaution of infection control in the delivery units was fairly enough and the majority of them had excellent score of practice. Recommendations: There should be active hospital infection control committees set up, combined with maternal death reviews, audits, training and feedback on infection rates.