Newborn screening is an important public health program that helps save the lives of many infants worldwide. The aim of this cross-sectional descriptive study was to examine the knowledge and attitudes of mothers regarding the newborn screening test in Jordan. A convenient sample of 301 mothers of newborns was interviewed to collect data, using structured questionnaires about their knowledge and attitudes regarding the newborn screening. Most mothers exhibited positive attitudes toward the newborn screening. However, their knowledge about it was only moderate; their knowledge levels contributed positively to their attitudes to the test. The mothers’ source of information about the test was a significant predictor for both their level of knowledge and attitudes toward the newborn screening. The healthcare providers, particularly nurses, were identified as the main source of mothers’ information in Jordan. The educative role of the health professionals is key and should be better activated to optimize the outcomes of early newborn screening. Changes in current practices regarding mothers’ education about NS is recommended to increase the knowledge and enhance attitude about NS among the mothers.