The area of Isernia was settled at least 700,000 years ago: the nearby site called Pineta has been cited in the magazine Science as the most ancient site where traces of use of fire by humans have been found.
Classical Aesernia was a city of Samnium, included within the territory of the Pentri tribe, situated in the valley of the Vulturnus (modern Volturno), on a small stream flowing into that river, and distant from Venafrum (modern Venafro). The Itinerary (in which the name is written "Serni") places it on the road from Aufidena to Bovianum, at the distance of from the former, and from the latter; but the former number is corrupt, as are the distances in the Tabula Peutingeriana.
The first mention of it in history occurs in 295 BCE, at which time it had already fallen into the hands of the Romans, together with the whole valley of the Vulturnus. After the complete subjugation of the Samnites, a colony, with Latin rights (colonia Latina) was settled there by the Romans in 264 BCE the city, a key communication center between southern Italy and the inner Regions. This colony is again mentioned in 209 BCE as one of the eighteen which remained faithful to Rome at the most trying period of the Second Punic War. During the Social War it adhered to the Roman cause, and was gallantly defended against the Samnite general Vettius Scato, by Marcus Claudius Marcellus, nor was it till after a long protracted siege that it was compelled by famine to surrender, 90 BCE. Henceforth it continued in the hands of the confederates; and at a later period of the contest afforded a shelter to the Samnite leader, Gaius Papius Mutilus, after his defeat by Lucius Cornelius Sulla. It even became for a time, after the successive fall of Corfinium (modern (Corfinio) and Bovianum, the headquarters of the Italic League. At this time it was evidently a place of importance and a strong fortress, but it was so severely punished for its defection by Sulla after the final defeat of the Samnites in 84 BCE, that Strabo speaks of it as in his time utterly deserted.
We learn, however, that a colony was sent there by Julius Caesar, and again by Augustus; but apparently with little success, on which account it was recolonized under Nero. It never, however, enjoyed the rank of a colony, but appears from inscriptions to have been a municipal town of some importance in the time of Trajan and the Antonines. To this period belong the remains of an aqueduct and a fine Roman bridge, still visible; while the lower parts of the modern walls present considerable portions of polygonal construction, which may be assigned either to the ancient Samnite city, or to the first Roman colony. The modern city is still the see of a bishop. The massively constructed podium now underlying the cathedral probably supported the Capitolium.
In the early 7th century AD, what are today the communes of Isernia as well as Bojano and Sepino were the places where Grimoald I of Benevento settled a group of Bulgars, seeking refuge from the Avars; the Bulgars were for many generations a distinctive part of the population, until finally assimilated in their Italian environment (see Bulgarians in Italy, Old Great Bulgaria#Bulgars in Southern Italy).
Even after the fall of the Western Roman Empire, Isernia has suffered destruction numerous times in history. Isernia was destroyed by the Saracens in 800, sacked by Markward of Anweiler, Count of Molise, in 1199, and set on fire in 1223 by the soldiers of Frederick II. In 1519 it was freed from feudal servitude by Charles V, Holy Roman Emperor and became a city in the Kingdom of Naples.
Earthquakes in 847, 1349, 1456 and 1805 caused massive destruction.
On the morning of September 10, 1943, during World War II, American planes launched their bombs from B-17 Flying Fortress planes over a crowded town on market day causing thousands of deaths. In the following weeks they came back twelve times without ever hitting their targets: the bridges of Isernia, Cardarelli and Santo Spirito, then built entirely of iron, towards the internal area. All the bridges were vital to the German retreat.
In 1970 Isernia became the capital of the homonymous province, created out of part of the province of Campobasso.