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The use of biostimulants offers a sustainable strategy to improve crop quality. This study assessed the effects of an arbuscular mycorrhizal fungi inoculum and an aqueous extract of Landoltia punctata on the growth and biochemical composition of lettuce (Lactuca sativa L. cv. ‘Dubáček’) under indoor conditions. Four variants were tested: control (C), mycorrhiza (M), L. punctata extract (L), and their combination (M+L), with biometric, physiological, and biochemical parameters evaluated. While biostimulant treatments did not affect above-ground biomass, L. punctata extract (L and M+L) significantly raised chlorophyll a (by 15.9 % and 16.0 %) and chlorophyll b (by 55.5 % and 42.8 %) compared to the control. The combined treatment (M+L) achieved the highest total phenolic content (254.28 mg/kg). All treated variants significantly reduced leaf nitrate content, with M and M+L being most effective (−35.1 % and −33.6 %). Amino acid metabolomic analysis showed that the extract is rich in γ-aminobutyric acid, valine, phenylalanine, tryptophan, and other proteinogenic amino acids that may drive its biostimulant effects. Microscopy confirmed successful root colonisation in mycorrhizal variants (58 % in M, 42 % in M+L). Overall, while biometric parameters remained unaffected, the applied biostimulants enhanced lettuce nutritional quality and physiological status by boosting photosynthetic pigments and phenolic compounds while lowering nitrate content, highlighting their potential for producing high-quality, safe crops.