BACKGROUND: Control of cutaneous leishmaniasis by public health systems in the Americas relies on case identification and treatment. Point-of-care diagnostics that can be performed by health workers within or near affected communities could effectively bring the health system to the resource-limited sites providing early diagnosis and treatment, reducing morbidity and the burden of disease. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was undertaken to evaluate the diagnostic test performance of Isothermal Recombinase Polymerase Amplification (RPA) targeting Leishmania kinetoplast DNA, coupled with a lateral flow (LF) immunochromatographic strip, in a field setting and a laboratory reference center. Minimally invasive swab and FTA filter paper samples were obtained by community health workers and highly trained technicians from ulcerated lesions of > 2 weeks' evolution from 118 patients' ≥ 2 years of age in the municipality of Tumaco, Nariño. Extracted DNA was processed by RPA-LF at a reference center or in a primary health facility in the field. Evaluation was based on a composite "gold standard" that included microscopy, culture, biopsy and real-time polymerase chain reaction detection of Leishmania 18S rDNA. Standard of care routine diagnostic tests were explored as comparators. Sensitivity and specificity of RPA-LF in the reference lab scenario were 87% (95%CI 74-94) and 86% (95%CI 74-97), respectively. In the field scenario, the sensitivity was 75% (95%CI 65-84) and specificity 89% (95%CI 78-99). Positive likelihood ratios in both scenarios were higher than 6 while negative likelihood ratios ranged to 0.2-0.3 supporting the usefulness of RPA-LF to rule-in and potentially to rule-out infection. CONCLUSIONS/SIGNIFICANCE: The low complexity requirements of RPA-LF combined with non-invasive sampling support the feasibility of its utilization by community health workers with the goal of strengthening the diagnostic capacity for cutaneous leishmaniasis in Colombia. TRIAL REGISTRATION: ClinicalTrials.gov NCT04500873.