Organic vegetable production in South Dakota faces challenges in nutrient management due to a short growing season that limits cover crop use. A two-year field study (2022–2024) was conducted at South Dakota State University’s Southeast Research Farm to evaluated the effects of living mulch species red clover (T. pratense L.), white clover (T. repens L.), and a white × kura clover hybrid (T. repens × T. ambiguum) and different tillage practices (no-tillage, no-tillage with fabric, tillage, and tillage with fabric) on soil biochemical properties under squash (Cucurbita maxima) and cabbage (Brassica oleracea L.) production. The results showed that living mulch integration improved soil moisture, microbial biomass carbon and nitrogen, water-extractable nitrogen, and permanganate oxidizable carbon, with species-specific responses across years. Red clover and the white × kura clover enhanced microbial biomass and labile carbon pools, while white clover improved soil moisture retention. No-tillage systems increased total carbon, nitrogen, phosphorus, potassium, and β-glucosidase activity compared to tilled systems. Synthesizing the findings, integrating leguminous living mulches with no-tillage management improved soil biochemical properties, nutrient cycling, and microbial activity, offering a sustainable approach for enhancing soil health and productivity in organic vegetable systems across South Dakota and similar temperate regions.