Over the centuries, suspicion has fallen on any number of potential poisoners, from Alexander's own wife and illegitimate half-brother to his generals and even the royal cup-bearer. But now a British historian believes he has finally solved the mystery: the killer of the greatest warlord in human history was nothing more than a humble mosquito.
Andrew Chugg, a respected authority on Alexander and author of a number of books on the subject, claims he has unearthed new evidence to suggest that the Macedonian conqueror died of malaria, contracted two weeks before his death while sailing in the marshes outside Babylon.
Mr Chugg bases his argument on the Ephemeredes, or Journal of Alexander, an ancient diary kept by one of the king's aides during his reign in the late 4th century BC. Critics have long questioned the authenticity of this source, claiming it was an elaborate later fabrication, but Mr Chugg believes he has now identified a number of "very sharp coincidences" that prove its veracity.
The author, he says, was Diognetus of Erythrae, a surveyor in Alexander's entourage, responsible for mapping out territories as they were conquered. The journal gives a detailed account of Alexander's last days, describing symptoms highly consistent with malaria.
"If you accept that the Ephemeredes is an accurate description of how Alexander died over a period of two weeks, then it is very difficult to believe the poisoning stories," said Mr Chugg, who will put his case in a forthcoming edition of the academic journal Ancient History Bulletin.
"It is known that the incubation period for malaria is between nine and 14 days, and about this time before he fell ill, we know that Alexander was sailing around the marshes outside Babylon inspecting flood defences. This is almost certainly where he was infected. Throughout history, and even to the present day, people have been contracting malaria in that particular area."
Mr Chugg claims he can also prove the authenticity of a second contemporary source: a commentary on Alexander's journal by another man who served under him, Ephippus of Olynthus, which further strengthens his case.
"It is difficult to imagine how anyone could have been fooled into compiling the commentary at such an early date if the journal itself had been fabricated," said Mr Chugg. "Ephippus of Olynthus, who had served Alexander in Egypt, was the author of an indisputably genuine book about Alexander's death."
Alexander, who died in Babylon in the summer of 323 BC, was last year the subject of a Hollywood "sword and sandals" epic, starring Colin Farrell and Angelina Jolie, as well as two biographies supporting the poisoning theory.
In Alexander The Great: The Death of a God, another British historian, Dr Paul Doherty, claimed that the murderer was the king's lieutenant and possible illegitimate half-brother Ptolemy.
"I believe that Alexander was killed with arsenic," said Dr Doherty. "There were lots of omens leading up to his death, suggesting that somebody was playing with his mind. There is also a tremendous amount of evidence suggesting that anyone who hinted that Alexander was killed came to a very nasty end. It all adds up to a murder plot."
But there was encouragement for Mr Chugg's theory from another of the subject's academic heavyweights, Professor Paul Cartledge of Cambridge University,
"This sounds very plausible to me," said Prof Cartledge, author of Alexander The Great: The Hunt for a New Past. "What Mr Chugg has shown is that there is evidence compatible with an argument that Alexander died of malaria, and I'm perfectly prepared to believe that."
Tutankhamun (c.1343-1324 BC)
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Distraught by the death of her lover Mark Antony, it is alleged that the Egyptian Queen Cleopatra invited an asp to sink its fangs into her breast. But historians say she was murdered, and the modern forensic finger has pointed to Octavian, the man who killed her lover.
Claudius (10 BC-54 AD)
Roman emperor Claudius may have died from a poisoned feather forced down his throat by a doctor, according to the historian Tacitus. His wife Agrippina supposedly instructed the doctor after she had failed with a plate of poisoned mushrooms.
Empress Catherine II 'The Great' (1729-1796)
According to myth, the German princess turned Russian Empress was crushed to death while trying to have sex with a horse. A more memorable alternative to the likely truth that she died of natural causes.
Napoleon Bonaparte (1769-1821)
Scientific findings in the 1960s suggested that Napoleon may have been poisoned. However, some historians blame the large quantity of arsenic found in his body on everything from the Emperor's drinking water to his hair cream.
Aline Nassif and Matthew Armitage
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