This study investigated the effects of salinity, ammonia, and stocking density on Nile tilapia (Oreochromis niloticus) fingerlings over a 74-days. In three separate experiments, fingerlings (initial weight 25 ± 2.4 g) were exposed to salinity levels (5, 10, 15, and 20 ppt), ammonia concentrations (0.01, 0.02, 0.1, and 0.2 mg/L), and stocking densities (10, 15, 20, and 25 fish per 96 L aquarium). Survival, growth performance, biochemical parameters, and gene expression changes were assessed. Salinity ≥ 15 ppt and ammonia ≥ 0.1 mg/L significantly impaired growth (final weight, weight gain, specific growth rate, and feed efficiency) and increased mortality rates, reaching 37% and 56% at 20 ppt salinity and 0.2 mg/L ammonia, respectively. Elevated salinity and ammonia also caused significant increases in the activities of ALT, AST, LDH enzymes, along with higher serum glucose levels, while disrupting serum protein and ion concentrations, indicating considerable metabolic and osmoregulatory disturbances. At the molecular level, the expression of the growth-promoting IGF-I gene was down-regulated, while inflammatory marker TNFα was up-regulated, suggesting compromised health. Stocking density had less pronounced effects, though densities ≥ 20 fish/aquarium led to reduced growth, altered biochemical markers, and gene expression changes compared to 10-15 fish/aquarium. These findings establish salinity and ammonia tolerance thresholds for tilapia fingerlings, emphasize optimal stocking density, and provide insights into the physiological and molecular responses to multifactorial stressors. The study contributes to sustainable management strategies for tilapia aquaculture under variable environmental conditions.