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To our knowledge, the bacterium that caused the Justinian Plague is <i>Yersinia pestis</i>, which is still found in the mountains of Tian Shan, which sit along the modern Kyrgyzstan, Kazakhstan, and China borders. This is a form of bubonic plague, very similar to the Black Death. While today the plague from this bacteria can be treated, periodic outbreaks would have likely devastated ancient populations without any known immunity. The spread of the plague could have had many reasons, but it could have been migrating travels from Central Asia, including possibly Huns, may have spread the plague. This was also a period of relatively cold winters and failed crops may have prompted migrations that brought the plague with travelers. The Silk Road was also an important trading network during the 6th century CE, suggesting travelers from Central Asia probably brought it to the eastern Mediterranean, where it then also spread into Europe from these Mediterranean ports. There is also evidence that the plague came through North Africa, as important trading ports connected Africa with eastern Asia. From Ethiopia, and into Egypt, the plague could have also expanded into the Middle East and into Europe. Both historical descriptions of the symptoms and excavated skeletons with evidence of the plague that date to this period suggest the plague, similar to the 14th century Black Death, was the culprit.

Procopius was a Byzantine historian and he reported an outbreak of the plague in 541 in the Egyptian port of Pelusium. Syriac ecclesiastical records also record the outbreak in Antioch and other regions of the eastern Mediterranean. During the high of the plague, Constantinople may have lost 5000-10,000 people per dayat the peak of the outbreak, although numbers are difficult to determine and the accuracy of reporting is disputed. Archaeological evidence from Germany and other northern European countries indicates that the disease also spread to these regions. While many towns and villages had already declined due to the collapse of the Roman Empire, other diseases, and famine, this also further devastated communities, perhaps prolonging northern Europe's Dark Age. Emperor Justinian, at the time, was busy financing the Hagia Sophia and carrying out his wars in the western Mediterranean. At that point, he was on the verge in retaking key parts of the western Mediterranean that would have resulted in the Byzantine Empire effectively reuniting lands from the Roman Empire. However, Justinian was forced to minimize his campaigns and likely his forces were weakened. Many crops failed as people were unable to attend to them and the cost of grain rose sharply. Wages also increased as labour supply became limited. The economy throughout the Mediterranean faced enormous strain, depleting the finances of the Byzantine Empire. Justinian also became infected with the plague but was able to survive. The Sasanian Empire, great rivals to the Byzantines who were based in Mesopotamia and Iran, were also affected, although we have fewer surviving records. Initially, they may have benefited, as they were able to make advances on the Byzantines in Armenia and in the Levant; however, this began a series of long-term wars with the Byzantines that likely depleted their forces over time, enabling the Sasanians to perhaps be more easily conquered in the 7th century by the rising Arabs.