Beneficial plant–microbe interaction for enhancing crop yield and quality is a sustainable way to achieve eco-friendly, desirable agricultural productions. The main objective of this experiment was to evaluate the individual and combined effects of an arbuscular mycorrhizal fungus (AMF) strain (Funneliformis mosseae) and a seaweed extract (SWE) derived from Ascophyllum nodosum, on the growth and physiological responses of lettuce (Lactuca sativa L.). Lettuce plants were inoculated with commercial AMF inoculum (5 g kg−1 soil), and SWE foliar application was done at three levels (0.5, 1.5, and 3 g L−1). The findings revealed that AMF along with SWE generated the greatest impact. In fact, co-application of AMF inoculation and 3 g L−1 SWE considerably enhanced root colonization, chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, and mineral content in the shoots and roots (N, P, K, Ca, Fe, Zn, and Mn content) of lettuce plants. This combination improved initial fluorescence (F0), photochemical efficiency of PSII (FV/Fm) and Y(NO) and total antioxidant activity (TAA), whereas the maximum fluorescence, (Fm) and Y(II), showed the highest increase in lettuce plants treated with AMF and 1.5 g L−1 SWE. Furthermore, AMF inoculation along with SWE, at concentrations 1.5 and 3 g L−1, considerably enhanced variable fluorescence (FV) and the activity of water decomposition in electron donor photosystem II (FV/F0). As a result of these findings, it can be stated that the co-application of AMF and SWE positively improves the growth and development of lettuce plants.