A door-to-door survey was organised in Cuenca, Ecuador, to determine the prevalence of COVID-19 infection and adherence of the population to COVID-19 preventive measures. A total of 2457 persons participated in the study; 584 (23.7%) reported having experienced at least one flu-like symptom since the onset of the pandemic. The maximum SARS-CoV-2 seroprevalence in Cuenca was 13.2% (CI: 12–14.6%) (IgM or IgG positive). Considering PCR confirmed infections, the prevalence was 11% (CI: 10–12.4%). There was no significant difference in seroprevalence between rural and urban areas. Participants aged 35–49 years old, living with a COVID-19 positive person, at least six people in a household, physical contact with someone outside the household, a contact with a person outside the home with flu-like symptoms, using public transport, and not having enough resources for living, significantly increased the odds for SARS-CoV-2 seropositivity. Overall, there was good adherence to COVID-19 preventive measures. Having known someone who tested positive for COVID-19, having a primary or secondary level of education, and having enough resources for living, significantly increased the odds for higher adherence. In conclusion, despite good overall adherence of the population of Cuenca with COVID-19 preventive measures, our study suggests high ongoing COVID-19 transmission in Cuenca, particularly in certain parishes. Prevention should not only focus on behavioural change, but on intensified testing strategies in demographical risk groups.