Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N-N bond formation reactions, including a glycine-utilizing hydrazine-forming enzyme, Tri28, and a nitrous acid-utilizing N-nitrosating enzyme, Tri17. This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N-N bond formation.