Background <p>Caring for preterm newborns imposes substantial psychological stress on mothers, often affecting their mental health and overall quality of life. Strengthening spiritual well-being, a key dimension of holistic health, may offer an effective coping resource. Addressing this need, the present study uniquely developed and evaluated an Islamic-based spiritual self-care training specifically tailored for mothers of preterm newborns, integrating structured spiritual practices (Adhkar, recitations, and guided reflection) rooted in Islamic teachings with psychological coping principles. This study aimed to investigate the effects of an Islamic-based spiritual self-care training program on stress and spiritual well-being among mothers of preterm newborns in Iran. The intervention was designed to incorporate Islamic spiritual principles and practices relevant to the maternal experience in this cultural context.</p> Methods <p>This quasi-experimental pretest–posttest study with sequential sampling included 95 mothers of preterm infants admitted to neonatal intensive care units. Participants were assigned to either an intervention group receiving Islamic-based spiritual self-care training or a control group receiving standard care. The program, conducted over six sessions, combined spiritual recitation, reflection, and self-regulation practices inspired by Islamic spirituality. Data were collected using the Miles and Funk Parental Stress Scale and the Ellison and Paloutzian Spiritual Well-being Scale and analysed using descriptive statistics and MANCOVA (SPSS v.20).</p> Results <p>The mean scores of post-tests spiritual well-being in the intervention and control groups were 108.00 ± 8.87 and 74.17 ± 13.00, respectively. The mean score of post-test maternal stress in the intervention and control groups was 46.74 ± 14.57 and 140.38 ± 10.00, respectively.</p> <p>The MANCOVA results demonstrated the impact of the intervention on both outcome variables: spiritual well-being and maternal stress (F = 110.547, <i>p</i> &lt; 0.001, partial η² = 0.720). Pairwise comparison analysis revealed that the intervention group had a higher mean post-test score for spiritual well-being (partial η² = 0.515) and a lower mean post-test score for maternal stress (partial η² = 0.679) after controlling for covariates (<i>P</i> &lt; 0.001).</p> Conclusions <p>The study demonstrates that Islamic-based spiritual self-care training significantly enhances mothers’ spiritual well-being and reduces stress, underscoring the value of culturally grounded, faith-integrated interventions in maternal mental health care. The unique contribution of this study lies in operationalizing Islamic spiritual practices into a reproducible, session-based intervention model. However, results should be interpreted with consideration of the quasi-experimental design and sequential sampling. Future randomized controlled trials in diverse Islamic settings are recommended to substantiate these findings and refine the integration of spiritual self-care into maternal support programs.</p> Trial registration <p>This study has been registered with the Iranian Registry of Clinical Trials (IRCT) under the registration code ‘IRCT20250530065981N1’ on 18/07/2025.</p>