Most terrestrial plants can establish symbiotic relationships with arbuscular mycorrhizal (AM) fungi, which increase the host plants’ resilience to pathogens. The effect of pre-inoculation with AM fungi as a bio-agent on lettuce (Lactuca sativa L.) plant resistance against Alternaria alternata RaSh3 leaf spot disease was investigated. The findings demonstrated that in A. alternata-infected plants, AM fungi could effectively colonize lettuce roots at a higher rate (100%) than in non-infected plants (91.66%). According to the disease assessment, lettuce plants pre-inoculated with AM and infected with A. alternata RaSh3 showed a 33.33 and 30.00% reduction in disease incidence and severity, respectively. During A. alternata RaSh3 infection, the primary growth responses, pigment fraction, proline, and carbohydrates of lettuce plants were reduced, accompanied by increases in oxidative stress markers [malondialdehyde (87%) and hydrogen peroxide (30.8%)]. Contrarily, AM-inoculated plants showed a significant increase in growth, photosynthetic pigments, osmolytes and enzymatic and non-enzymatic antioxidant enzymes either in A. alternata RaSh3-infected or non-infected ones. Overall, our results highlight the significance of AM fungi in alleviating infection symptoms by increasing proline (13%), flavonoids (28.3%), and phenolic compounds (44.7%). Moreover, a boost in the enzymatic status (phosphatases, antioxidants, and phenylalanine ammonia-lyase) was detected in A. alternata RaSh3-infected plants due to AM inoculation, proving the essential role of its inoculation in increasing plant resistance against A. alternata RaSh3. Finally, this experiment has proved the sustainable defense strategy of mycorrhizal symbiosis as a new bio-agent for the biological control of A. alternata in lettuce plants