<b><i>Aim:</i></b> The 2015 recommendation of the International Liaison Committee on Resuscitation of no routine tracheal suctioning in non-vigorous neonates born through meconium-stained amniotic fluid (MSAF) was based on very low certainty of evidence (CoE) necessitating ongoing monitoring. The aim of this systematic review was to perform a meta-analysis of observational studies comparing the effect of implementing immediate resuscitation without routine tracheal suctioning versus with routine suctioning in neonates born through MSAF. <b><i>Methods:</i></b> MEDLINE, Embase, CENTRAL, and Web of Science were searched. Observational studies with a before-and-after design were included. Two authors extracted data independently. CoE based on GRADE recommendations was performed. <b><i>Results:</i></b> 13 studies were included. Clinical benefit or harm could not be excluded for the composite primary outcome of mortality or requirement of extracorporeal membranous oxygenation (ECMO) (relative risk, 95% confidence interval: 0.74 [0.47–1.17]), and mortality (0.68 [0.42–1.11]). “Routine tracheal suctioning” epoch had possibly lesser risk of meconium aspiration syndrome (MAS) when compared to “no routine tracheal suctioning” epoch (0.68 [0.47–0.99]). “Routine tracheal suctioning” epoch also possibly had a lower risk of hospital admission for respiratory symptoms, requirement of non-invasive respiratory support, invasive mechanical ventilation, surfactant treatment, air leak, and low-flow oxygen therapy. Clinical benefit or harm could not be excluded for the outcome of mortality or ECMO among those diagnosed with MAS (1.09 [0.86–1.39]), but “routine tracheal suctioning” was possibly associated with lower risk of respiratory morbidities among those diagnosed with MAS. The CoE was very low for most of the outcomes evaluated. <b><i>Conclusions:</i></b> Due to the very low CoE for the outcomes evaluated, no definitive conclusions can be drawn warranting the need for additional studies.