Background: Strongyloides stercoralis is a neglected soil-transmitted helminth endemic in tropical and subtropical regions. This study aimed to determine the prevalence, risk factors, and spatial distribution of S. stercoralis infection in rural villages of Khon Kaen Province, Northeastern Thailand. Methods: A cross-sectional survey was conducted among 260 participants from 201 households across five randomly selected villages. Stool samples were analysed using agar plate culture and formalin-ethyl acetate concentration techniques, and soil samples were assessed for S. stercoralis DNA by PCR and physicochemical properties including pH, porosity, bulk density, moisture, organic matter, and nitrate. Results: The prevalence of S. stercoralis infection was 16.15%, with agar plate culture detecting 41 cases (15.77%) compared to 12 cases (4.62%) by formalin-ethyl acetate concentration techniques. The highest prevalence was observed in Norng Thungmon village (31.80%). Risk factors included male gender, older age, being a head of household, living in homes with earthen floors, walking barefoot, and proximity to water sources. Soil samples from 169 households revealed an S. stercoralis detection rate of 8.3% via PCR. Positive soil samples were predominantly from Norng Huachang and Don Du villages. Spatial analysis identified significant clustering of infections within high-risk areas. Soil physicochemical properties, including pH, moisture, and organic matter, were measured, though no significant correlations were found with infection rates. Conclusion: In conclusion, S. stercoralis remains a significant health concern but is neglected in rural Khon Kaen, with identifiable human and environmental risk factors. Enhanced health education, improved sanitation, and targeted interventions are essential for reducing transmission in endemic areas.