Azoxy compounds are a distinctive group of bioactive secondary metabolites characterized by a unique RN═N(O)R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by P8-A2. Here, we cloned and confirmed the putative biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, J1074 and M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knockout using CRISPR-Cas9 base editing in the native producer, P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to an azo compound followed by further oxygenation. Enzymes involved in modifications of the precursor could be postulated based on their predicted function and the intermediates identified in the knockout strains. Moreover, the distribution of the azoxy biosynthetic gene clusters across spp. genomes is explored, highlighting the presence of these clusters in over 20% of the spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A-like clusters are widely distributed across the phylogenetic tree.