The Portuguese had reportedly been considering a January move for the Argentine, who has netted 13 goals for the Naples outfit this season and bagged 48 league goals in three years under Mourinho's stewardship at Real Madrid.
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Asked whether there was any truth in a bid for Higuain, Mourinho told reporters: "No." Has there been any bid? "No." Will there be a bid: "No."
The 50-year-old then added: "Napoli want to win the Scudetto, they want to win the Europa League. I don't believe they're interested in selling Higuain. [He is] a player I know we cannot get."
During the Blues' 3-0 win over Southampton on Wednesday, Juan Mata showed his frustration at being substituted, with Mourinho later implying that he would not stand in the Spaniard's way should he ask to leave Stamford Bridge.
But the former Inter manager believes the playmaker remains committed to the club and is keen to retain his services despite suggesting he could depart in January.
"[Mata trained] in a fantastic way," Mourinho added. "I think he is focused on [when I said] 'I don't want him to leave'."