Different fungal species and the bacterium Pantoea ananatis (Pa) have been reported as etiological agents of Phaeosphaeria leaf spot (PLS) disease. This work aimed at using molecular identification of the fungi and bacteria occurring in PLS and its etiology. Genomic DNAs from (i) pool of each of the four stages of PLS lesions; (ii) bacteria and fungi isolated from lesions of natural PLS (NPLS) and artificial injuries (AI); (iii) fungi isolated from lesions obtained from plants inoculated with Pa in greenhouse (GH) were used in PCR with universal primers for bacteria and fungal rRNA genes, and species-specific primers for Pa. Bacterial amplicons were observed at all stages of lesions and fungal amplicons in stages 3 and 4. Bacterial amplicons of pooled NPLS lesions were from Pa while fungal amplicons were from Phaeosphaeria sp. and Phoma sp. Bacteria from NPLS, GH and AI lesions were identified as Pa, Pa and Bacillus subtilis, respectively, while the fungi were Epicoccum nigrum, Leptosphaeria sacchari, Cochliobolus geniculatus, Pithomyces chartarum, Alternaria alternata, A. ricini, Gibberella intricans, G. fujikuroi, Phaeosphaeria sp., P. avenaria, Phoma sp., Phyllosticta sp., Sarocladium strictum, Glomerella graminicola, and Cochliobolus heterostrophus. Symptoms of PLS decreased by 90% with the use of oxytetracycline in foliar treatment of maize plants in the field, and its addition to culture medium completely inhibited the growth of Pa. The results strongly show that Pa is the causal agent of PLS disease and different species of opportunistic fungi appear late in the necrotic stages of lesions caused by Pa.