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This study compared microbiological and chemical methods used in astaxanthin extraction from the exoskeleton of the shrimp species Penaeus japonicusand Penaeus semisulcatus. The microbiological method was performed using Saccharomyces cerevisiae(bakery yeast) or Lactobacillus acidophilus(from yogurt), followed by solvent extraction with hexane and acetone at different ratios (1:1, 1:2, and 1:3). The chemical method was performed traditionally using hexane. The highest astaxanthin yield from P. japonicusexoskeleton was obtained using either S. cerevisiaeor L. acidophilusfollowed by solvent extraction with hexane and acetone at a ratio of 1:1 (8.5 and 8.1 mg/g waste, respectively) as well as by the chemical method (8.4 mg/g waste). Likewise, the highest astaxanthin yield from P. semisulcatusexoskeleton was obtained using either S. cerevisiaeor L. acidophilusfollowed by solvent extraction with hexane and acetone at a ratio of 1:1 (3.0 and 4.1 mg/g waste, respectively) as well as by the chemical method (3.2 mg/g waste). The values obtained from P. semisulcatusexoskeleton were considerably lower than those attained from P. japonicusexoskeleton. In addition, the nuclear magnetic resonance (C-NMR) analysis confirmed that astaxanthin was the main carotenoid present in the extract. In conclusion, the pretreatment of exoskeleton wastes of P. japonicususing S. cerevisiaefollowed by solvent extraction with hexane and acetone at a ratio of 1:1 as well as the classical chemical treatment led to the highest astaxanthin content.