Tomato spoilage is a major concept of annual revenue wastage in some countries in Africa. In the Northern part of Nigeria, tomato fruits glut has been major problem due to the activities of microorganisms during transportation and storage before sales to the final consumers. This has led to the spoilage of tones of this important fresh produce over decades. Spoilage was initiated in fresh tomato fruit by conditioning them to semi-humid environment and storage at 27±2°C for 14days during which the microbiological and chemical evaluation was examined. Seven organisms were isolated from the decomposed tomato fruits and characterized using genomic DNA extraction, polymerase chain amplification of rRNA and sequence determination. The bacterial isolates were identified on the basis of the 16S rDNA as Bacillius laterosporus, Lactobacillus salivarius and Proteus vulgaris while, yeasts and filamentous fungi were identified on the basis of 26S rDNA as Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Rhodotorula musilaginosa, Aspergillus flavus, Aspergillus niger and Fusarium oxysporum. Three of the identified organisms namely; Bacillus subtilis, Aspergillus niger and Saccharomyces cerevisiae were isolated from the decomposed and control samples. The pH of the decomposing samples showed significant (p<0.05) increase with concomitant decrease in titratable acidity after storage for 8 days. There was significant (p<0.05) increase in the vitamin K of decomposed tomato (8.76 – 9.45 mg/100g) at the end of 14 days storage when compared control (1.09mg/100g). The carotenoid fractions of the decomposed samples decreased significantly (p<0.05) except malvidin which increased significantly (7.99 – 9.02mg/100g) compared with the values of fresh sample (4.96mg/100g) that was used as control.