Sardines contain high levels of histidine within their muscles which is converted to histamine (HIS) by histidine-decarboxylase forming bacteria. Our investigation aimed to study the effect of storage time and temperature on the HIS levels of different muscular areas of sardine. Thirty fresh sardine samples were divided into two groups (n=15). Group (1) was kept at 0 ºC while group (2) was kept at 21 ºC. All fish were examined for freshness at time intervals of 0-, 6-, 12-, 18-, and 24-hr post fishing using Quality Index Method (QIM) and European grading scheme (EU) grading scheme. At the same time intervals, muscle samples were collected from the abdominal, dorsal, and tail regions for histamine measurement, bacterial isolation and identification as well as for histopathological examination. Our results revealed that the fish freshness was decreased by the storage time and temperature. Moreover, both bacterial count and histamine levels in different muscular areas were increased also throughout the storage time and temperature. Higher levels were observed in the abdominal muscle more than tail muscle and dorsal muscles. Enterobacter aerogenes, Enterobacter cloacae complex, Lelliottia amnigena-1 (Enterobacter amnigena-1) were identified with Vitek2 which gave 99 % confidence of identification, additionally, we localized gram-negative bacteria throughout the muscle tissue, primarily in the intermuscular region, around the blood vessels, and in the myofibrils. We concluded that during storage, there was a positive relationship between the HIS levels and the histamine-forming bacteria (HFB) numbers, sensory scores, and microscopic lesion scores. Also, the identified isolated bacteria differ from the previously recorded isolates in sardines and its related species at other localities; so the bacterial isolates might depend on the fishing locality.