BACKGROUND: To achieve improved health outcomes of HIV positive patients receiving ART services, quality laboratory services must form an essential part of the services provided. The aim of this study was to compare service quality by assessing the level of adherence to quality system essentials (QSEs) in laboratory services delivered by public and private health institutions in Southern Nigeria. METHODS: This was an analytical cross-sectional study conducted among 50 health facilities’ laboratories in 5 Southern States (Akwa Ibom, Anambra, Cross River, Edo and Rivers) of Nigeria. Randomly selected sample of secondary health facilities’ laboratories (ten per state, and a total of 25 public and 25 private health facilities) receiving equal level of support from the same USAID/PEPFAR implementing partner and had been providing ART services to clients for a minimum of one year, were included in the study. Quarterly Internal quality audit was conducted in the ART Laboratory section of the selected health facilities spanning July 2015 to September 2016. 200 audit reports were checked, cleaned, and analyzed using SPSS version 23. We analyzed changes in mean performance scores over time across 6 quality management essentials using Repeated Measures ANOVA. Results were considered significant at P<0.05. RESULTS: The result of the study showed that the private health facilities laboratory achieved a significantly higher improvement in Facility and Safety score over time (p=0.019) compared to public health facilities. Overall, temporal improvements were recorded in all facilities in three out of the six QSEs (document and record, p=0.045; organization and personnel, p=0.020; equipment, p<0.001) and total laboratory quality score (p=0.004). But there was no significant quarterly difference in performances on QSEs between public and private health facilities laboratories. CONCLUSION: Our findings indicate that despite receiving the same level of external support, private hospital laboratories adhere better to the quality standards on Facility and safety than public hospital laboratories in Southern Nigeria.