Background: Childhood Disease prevention practices (DPP) by day-care/preschools caregivers are essential to the wellbeing of enrolled children. We assessed DPP such as hand hygiene, feeding, nutrition, handling sick children and pre-employment medical screening in day-cares /pre-schools and the factors associated with DPP.Methods: The cross-sectional study involved 799 pre-school caregivers (teachers and minders/nannies). Ten settlements were selected from five randomly selected metropolitan Local Government Areas in Oyo state, Nigeria. Data collected on DPP was mainly graduated on 3-point Likert like scale: ‘always responses’- allotted 2 points; ‘sometimes’- 1 point; and ‘never’- zero. Using a significance level (p value) set at 5% and higher R-squared values, associations between DPPs and explanatory variables were tested).Results: Mean age of respondents was 33.7±9.5 years. Majority, 594 (74.3%) did not have pre-employment childcare training. Self-reported hand hygiene was highest for stool moments. Most 456 (56.3%) reported formula feeds for 1-6 months and mainly staples for older children. Aggregated DPP score (80) was dichotomized using mean childcare DPP score of 24±4.5. Slightly more than half of the respondents, 453 (56.7%) had inadequate DPP. Formal childcare training p<0.001, current job specification p=0.02 and knowledge of VPD p=0.004 were associated with DPP index. Some predictors of adequate childcare DPP include facility registration status (OR=2.19, 95%CI=1.05-4.56); respondents who had childcare training (OR=1.52, 95%CI=1.083-2.144); affiliation with health (OR=2.0, 95%CI=1.227-3.262).Conclusions: This study highlights childcare DPP within the day-cares/pre-schools and provides evidence for tailored training interventions and monitoring of the facilities.