Di Francesco led the Neroverde into Serie A for the first time in their history - an achievement for which he was named Serie B coach of the year - only to be sacked in January after a run of poor results.
Former Parma, Genoa and Palermo boss Malesani, 59, was installed but oversaw five straight defeats as Sassuolo sank to the bottom of the table, where they are currently four points from safety.
A statement published on sassuolocalcio.it read: "Sassuolo announce they have relieved Alberto Malesani of his duties as first-team coach.
"He deserves thanks for his professionalism and commitment during this difficult period of the season.
"The technical guidance of the first team will be entrusted to Eusebio Di Francesco."
The 44-year-old Di Francesco - a former midfielder with Roma and Perugia - on Monday won the Panchina d'Argento award for guiding Sassuolo into Serie A as second-tier champions in 2013.
He told the Gazzetta dello Sport: "It was an exciting season and we out-performed some very important teams. It is an award I'll share with my staff and with the players, who were outstanding."
He resumes his career on the Sassuolo bench against fellow Emilian club Bologna on Sunday before returning to the Stadio Citta del Tricolore to host relegation rivals Catania on March 16.