This study aimed to evaluate the influence of extrusion temperature and moisture incorporation procedure on nutrient digestibility of diets fed to barramundi (Lates calcarifer). A single premix was processed under six contrasted extrusion processing conditions, with extruder barrel temperatures set at 100 °C and 110 °C, and liquid water either incorporated in the preconditioner, in the barrel, or in both. In a subsequent 33-day feeding trial, grow-out fish (758.3 ± 24.5 g) were fed the 6 diets and apparent digestibility coefficients (ADC) were assessed for 42 fatty acids, 17 amino acids, protein, lipids and energy. Digestibility of protein (92.3–93.7%), lipid (84.1–89.4%), and energy (69.4–75.2%) also remained unaffected by extrusion settings (P > 0.05). However, lower extrusion temperature and moisture incorporation in both preconditioner and barrel resulted in higher digestibility of poly-unsaturated fatty acids (PUFA), long-chain poly-unsaturated fatty acids (LC-PUFA), omega-3, omega-6 and 22:6 docosahexaenoic n-3 (DHA). Moreover, lower extrusion temperature significantly improved ADCs of essential amino acids, except lysine, histidine, and threonine (P < 0.05). The results showed no significant impact (P > 0.05) of the tested extrusion parameters on fish growth (TGC: 2.11 ± 0.17), feed intake, or feed conversion performance (FCR: 1.67 ± 0.13). The study unveils diverse responses to extrusion temperature and moisture levels regarding amino acid and fatty acid digestibility. These findings highlight the advantages of preconditioning during the extrusion process to enhance fatty acid digestibility.