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Simple Summary: Diversification of cultured fish species is one key measure to promote the sustainable development of marine aquaculture. Mullets (Mugilidae) present great potential due to their eurythermal, euryhaline, and low-trophic nature. However, there are many similar mullet species, and their physiological differences and optimal culture conditions are quite unknown, mainly when reared under intensive systems. In this sense, increasing the knowledge about changes in their digestive biochemistry related to feeding strategies is essential for a sustainable and cost-effective culture of these species. For these reasons, the present study aimed to characterize the digestive biochemistry of two mullet species (Chelon labrosus and Liza aurata) and to evaluate how it is affected when fish are fed using two different feeding frequencies: one or three times per day. In addition, changes in the potential bioavailability of nutrients determined by the two feeding patterns were assessed using digestive simulations performed in vitro. The results demonstrated the convenience of a feeding pattern of three meals per day instead of one. Moreover, the results evidenced that although Chelon labrosus and Liza aurata are species placed in a similar trophic level, their digestive response to feeding patterns, as well as body composition, present differences when fish are reared under the same conditions. This suggests that such interspecific variation must be considered when fish are reared under intensive conditions. Mullets (Mugilidae) present significant potential for sustainable aquaculture diversification due to their eurythermal, euryhaline, and low-trophic nature. However, the physiological differences and optimal cultured conditions among the diverse mullet species are quite unknown. For these reasons, the present study aimed to address two main objectives: (1) to characterize the differences in digestive biochemistry, somatic indexes, and body composition between two mullet species (Liza aurata and Chelon labrosus); and (2) to evaluate the interactions of two different feeding frequencies (one against three meals per day) on the above-mentioned parameters, and also on the potential bioavailability of nutrients determined using in vitro assays. The results evidenced higher protease and amylase activities for Chelon labrosus than for Liza aurata, while the latter species presented a higher percentage of eviscerated weight and muscle lipids. Furthermore, the results from in vitro assays supported the higher enzyme activity of Chelon labrosus by an observed increase in the release of amino acids and reducing sugars measured for this species. Regarding feeding patterns, the results of the in vitro assays simulating enzyme: substrate (E:S) ratios corresponding to one or three meals per day point to a clear increase of nutrient bioavailability when the daily ration is split into several meals. The present results improve the physiological knowledge of mullet species and define criteria to develop better management protocols by producers. [ABSTRACT FROM AUTHOR]