Magnesium may reduce carotenoid bioavailability by forming insoluble complexes with bile salts/fatty acids, inhibiting micelle formation. Here, we investigated whether altering bile/pancreatin concentration influenced potential negative effects of magnesium on carotenoid bioaccessibility. Spinach (4 g) was digested in vitro with added magnesium (0, 200, 400 mg/L) and canola oil/coffee creamer, at varying bile extract (1 or 8 mM) and pancreatin (100 or 990 mg/L) concentrations. Bioaccessibility was determined for β-carotene, lutein, and total carotenoids via HPLC. Additionally, lipolysis, particle size, and zeta potential of the micellar fractions were investigated. Increasing magnesium concentrations negatively affected carotenoid bioaccessibility (p < 0.001), lipolysis, particle size and zeta potential. The impact of magnesium on carotenoid bioaccessibility was modulated mainly by bile concentration, with samples digested with 1 mM of bile being more susceptible to inhibitory effects of magnesium than those digested with 8 mM (p < 0.001). Thus, magnesium was found to potentially interfere with carotenoid bioaccessibility at various physiologically plausible conditions.