There has been an increase in the prevalence of gram-positive bacteremia in neonates in the last two decades. However, as a consequence of better care, there has been an increase in the survival of premature neonates. Coagulase-negative staphylococci (CoNS) is the most prevalent bacteria, responsible for up to 60% of late-onset sepsis (LOS). Daptomycin, a lipopeptide antimicrobial agent, is active against CoNS. This was an observational, retrospective case series study carried out in the Pediatric Hospital of King Saud Medical City, Riyadh, Saudi Arabia. The medical records of 21 neonates, aged 0–28 days, who were treated in Neonatal Intensive Care Unit (NICU) with intravenous daptomycin as monotherapy or combination therapy for at least 4 days for proven gram-positive infection between June 2019 to July 2020, were included. The median gestational and chronological age were 27 weeks and 5 days, respectively. The most frequent diagnosis in neonates was infective endocarditis (42.9%). Of the 21 patients who received daptomycin therapy, 13 (62%) recovered and 8 died. The clinical cure rate was higher in Staphylococcus hominis (100%) and in patients who received 6 mg/kg/dose twice daily (62.5%). The mean of aspartate aminotransferase significantly elevated after starting daptomycin (p = 0.048). However, no muscular or neurological toxicity of daptomycin was documented in any of the cases. Overall, daptomycin was well tolerated, even with long-term treatment.