Chlorophyll fluorescence (CF), growth parameters, phytochemical contents [proline, chlorophyll, ascorbic acid, total phenol content (TPC), total flavonoid content (TFC)], and antioxidant activities were investigated in lettuce (Lactuca sativa L.) seedlings grown under different sodium chloride (NaCl) concentrations (0, 50, 100, 200, 300, and 400 mM) in a controlled environment for eight days. The parameters were evaluated at two days intervals. Almost of the CF and growth parameters as well as phytochemicals were significantly affected by both NaCl concentrations and progressive treatment schedule. The maximum quantum yield (Fv/Fm), effective quantum yield of photochemical energy conversion in PSII [Y(PSII)], coefficient of photochemical quenching (qP), coefficient of non-photochemical quenching (qN), and ratio of fluorescence decline (Rfd) showed decrements only at the highest saline concentration (400 mM), whereas the quantum yield of non-regulated energy dissipation in PSII [Y(NO)] exhibited a dissipation trend. All the growth parameters decreased with increasing NaCl concentrations, showing the highest decrease (~8 fold) in shoot fresh weight, compared to control seedlings. Proline significantly increased with increasing NaCl concentration and treatment time. Other phytochemicals decreased with the increase in NaCl concentration and reached their lowest at 400 mM. Overall, the results showed major changes in all parameters when the seedlings were grown at a NaCl concentration of 400 mM. The present findings will be useful for understanding the differential effect of NaCl concentrations in lettuce seedlings, and also might be useful to optimize the NaCl concentrations in other crops grown in controlled environmental conditions.