Abstract: This study aimed to investigate the underlying mechanisms and efficacy of micro-nano ozone water technology in alleviating continuous cropping obstacles in facility-grown lettuce. Peat substrate previously used for two consecutive lettuce cropping cycles was employed in a third cultivation cycle. Four distinct experimental treatments were applied: continuous cropping substrate irrigated with clear water (T1), continuous cropping substrate irrigated with ordinary ozone water (T2, ozone concentration of 2 mg/L), continuous cropping substrate irrigated with micro-nano ozone water (T3, ozone concentration of 2 mg/L), and fresh substrate irrigated with clear water acting as the control group (CK). The experiment comprehensively compared lettuce growth, physiological parameters, root morphology, antioxidant capacity, and substrate health across these treatments.The results demonstrated that continuous irrigation with clear water (T1) significantly inhibited lettuce growth and physiological development. Compared to the CK group, the plant height, spreading width, and stem diameter of lettuce in the T1 group decreased by 6.47%, 6.19%, and 7.30% (P<0.05), respectively, while the fresh weight declined by 5.47%. Continuous cropping severely impaired root architecture, reducing total root length, root surface area, root volume, average root diameter, root tip number, and root fork number by 58.50%, 56.99%, 56.11%, 16.98%, 49.85%, and 18.58% (P<0.05), respectively. Furthermore, root fresh weight and dry weight decreased by 31.08% and 51.79%. At the physiological level, the T1 treatment disrupted the stability of the photosynthetic apparatus, the photosynthetic electron transport rate declined, and lettuce quality indicators deteriorated. Continuous cropping also deteriorated leaf nutritional quality and antioxidant defense systems. Soluble sugar and ascorbic acid contents dropped by 32.55% and 16.67%, respectively, while nitrate content increased by 22.72%. Activities of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and total antioxidant capacity (T-AOC), plummeted by 73.21%, 68.59%, 27.36%, and 66.47% (P < 0.05), respectively. This was accompanied by a 51.73% increase in malondialdehyde (MDA) and a 118.30% increase in proline (Pro). Both ozone water treatments mitigated these adverse effects, with the micro-nano ozone water treatment (T3) proving most effective: it enhanced root architecture by increasing total root length, surface area, volume, tip number, and fork count by 133.18%, 108.73%, 95.65%, 55.67%, and 19.35% (P<0.05), respectively, relative to T1; furthermore, T3 reduced fungal abundance in the substrate by 87.83%, increased urease activity by 16.48%, and ultimately improved lettuce fresh weight by 6.73% and soluble sugar content by 41.14% compared to T1. These comprehensive findings demonstrate that ozone water effectively alleviates continuous cropping obstacles by improving the rhizosphere microenvironment, optimizing the microbial community structure, and promoting nutrient transformation. Micro-nano ozone water (T3) is particularly effective in optimizing root system development, repairing the photosynthetic electron transport chain, and enhancing the plant’s antioxidant capacity, thereby providing a highly promising theoretical foundation and practical strategy for sustainable management practices in facility agriculture.