The internet can be used as an alternative method in infant care education for mothers of preterm infants. This study was conducted to examine the effect of the Web-based infant care training program given to mothers of preterm infants on the self-confidence levels of mothers. This nonrandomized controlled study with a posttest design was conducted in Ministry of Health training and research hospital and university hospital. The study group comprised 84 mothers and their infants (intervention group: N = 42; control group: N = 42). Mothers with preterm infants in the intervention group participated in the Web-based program. The self-confidence of the mothers was evaluated before and after the training. The mothers’ Pharis self-confidence scale mean scores were higher than the control group, but there was no significant difference. Web-based education program can be an alternative method that can be used in the education of mothers with preterm infants.