Fungi associated with fruit rot of tomato were isolated and the effects of storage temperature and relative humidity on rot development were investigated using standard Mycological techniques. The predominant fungi isolated were Aspergillus niger, Alternaria solani and A. flavus with 27.5%, 20.6% and 20.10% frequencies of occurence respectively. The least frequently encountered fungus was Mucur racemosus with 7.40% frequency of occurence. Pathogenicity test revealed that all the isolated fungi were pathogenic on healthy tomato fruits and have the potential to cause fungal diseases on tomato. The relative humidity for maximum rot development ranged from 70-90%. Tomato fruits stored well at 0-10 °C and rather poorly at 20-30 °C. Fruits stored at 35 °C for five days were found to be completely rotten. The optimum RH for tomato storage ranged between 60 to 90%. Manipulation of storage environmental factors such as temperature and relative humidity are important in controlling microbial infection of tomato fruit especially in tropical countries such as Nigeria.