Background Universal HIV pediatric screening offered at postnatal points of care (PPOC) is an entry point for early infant diagnosis (EID). We assessed the parents' acceptability of this approach in Abidjan, Côte d'Ivoire. Methods In this cross-sectional study, trained counselors offered systematic HIV screening to all children aged 6–26 weeks attending PPOC in three community health centers with existing access to HAART during 2008, as well as their parents/caregivers. HIV-testing acceptability was measured for parents and children; rapid HIV tests were used for parents. Both parents' consent was required according to the Ivorian Ethical Committee to perform a HIV test on HIV-exposed children. Free HIV care was offered to those who were diagnosed HIV-infected. Findings We provided 3,013 HIV tests for infants and their 2,986 mothers. While 1,731 mothers (58%) accepted the principle of EID, only 447 infants had formal parental consent 15%; 95% confidence interval (CI): [14%–16%]. Overall, 1,817 mothers (61%) accepted to test for HIV, of whom 81 were HIV-infected (4.5%; 95% CI: [3.5%–5.4%]). Among the 81 HIV-exposed children, 42 (52%) had provided parental consent and were tested: five were HIV-infected (11.9%; 95% CI: [2.1%–21.7%]). Only 46 fathers (2%) came to diagnose their child. Parental acceptance of EID was strongly correlated with prenatal self-reported HIV status: HIV-infected mothers were six times more likely to provide EID parental acceptance than mothers reporting unknown or negative prenatal HIV status (aOR: 5.9; 95% CI: [3.3–10.6], p = 0.0001). Conclusions Although the principle of EID was moderately accepted by mothers, fathers' acceptance rate remained very low. Routine HIV screening of all infants was inefficient for EID at a community level in Abidjan in 2008. Our results suggest the need of focusing on increasing the PMTCT coverage, involving fathers and tracing children issued from PMTCT programs in low HIV prevalence countries.