Open defaecation (OD) has been strongly linked to high prevalence of cholera and other sanitation-related diseases. This study was conducted to assess the effect of community-led total sanitation (CLTS) on sanitation facilities and the bacteriological quality of natural water sources in Cross River State. The study design was a community-based randomized controlled trial. A multi-stage sampling technique was used in selecting respondents. Data were collected from 744 respondents at pre- and post-intervention. A purposive sampling method was used in sampling natural water sources. Observational checklist and sterilized sample bottles were used in data collection. Data were analyzed using Statistical Product and Service Solution (SPSS version 24). Results were presented as frequencies, percentages and tables. The findings showed that respondents indicated that the major challenges of owning toilets (pre-intervention) were cost of constructing one 244 (32.8%), and distance of the toilet location 259 (34.8%) as major causes of OD practice. The same challenges were reported at post-intervention; as cost of construction 270 (36.3%) and distance of the toilet location 283 (38.0%). At pre-intervention, practice of OD was 253 (34.0%) among respondents who had no access to toilets but later reduced to 102 (13.7%) at post-intervention due to improved access to toilets. This infers that there was a statistically significant association between access to toilet and the reduction of OD practices due to the CLTS intervention. The test of hypothesis at both pre- (ϰ2 = 287.749; df = 1; P-value = 0.001) and post-intervention (ϰ2 = 8.334; df = 1; P-value = 0.001) was significant (P < 0.05). At pre-intervention, respondents with access to improved water sources were 119 (16.0%) which was improved to 569 (76.5%) at post-intervention, indicating that there was a statistically significant association between access to improved water sources and the reduction of sanitation-related diseases among respondents due to the CLTS intervention. Bacterial analysis showed that the total coliform and faecal coliform counts for all water samples exceeded the World Health Organisation (WHO) and the Nigerian Standard for Drinking Water Quality (NSDWQ) limits. Based on the findings, the CLTS intervention was effective in increasing knowledge of OD practice, changing wrong perceptions about OD and improving toilet ownership and maintenance. To scale-up the gains of this study, continued commitments to the eradication of OD practices from community members, community leaders, governments at all levels and NGOs is crucial.