Melanin is a complex biopolymer with antioxidative, UV-protective, and antimicrobial properties. Melanin is also of interest for bioengineering applications in healthcare. Its production has been frequently observed in several bacteria and higher organisms under specific culture conditions through genetic engineering and chemical mutagenesis. Interestingly, l-DOPA, a precursor to the neurotransmitter dopamine and an effective anti-Parkinsonian drug, has also been frequently observed, at lower levels, along with melanin in the culture of Bacillus thuringiensis, despite the bacterium lacking l-DOPA-producing tyrosinase sequences in the genome. The present study aims to predict the possible l-DOPA-producing enzyme and characterize the melanin biosynthesis pathway in B. thuringiensis var. israelensis MB-24, a strain derived by NTG mutagenesis of entomopathogenic B. thuringiensis var. israelensis B-17. Using metabolomics, we identified the key metabolites involved in melanin production. We also predicted the probable enzyme involved in l-DOPA production through conserved domain search. Sequencing the homogentisate 1,2-dioxygenase (hmgA) gene of MB-24 showed large deletions, suggesting that melanin synthesis may result from accumulated homogentisate in the HGA (Homogentisic acid) pathway. We expressed 4-hydroxyphenyl pyruvate dioxygenase from B. thuringiensis var. israelensis B-17 and characterized the melanin produced by this enzyme through FT-IR (Fourier-Transform Infrared Spectroscopy). The FT-IR analysis further verified that B. thuringiensis var. israelensis MB24 mostly produced pyomelanin. In conclusion, pyomelanin production in B. thuringiensis var. israelensis MB-24 is driven by the homogentisate pathway due to the inability of the mutant bacterium MB-24 to express functional homogentisate 1,2 dioxygenase. These findings inform future industrial and pharmaceutical applications of melanin biosynthesis.